Antibodies against TIM3 and uses thereof

ABSTRACT

Provided herein are antibodies, or antigen-binding portions thereof, that bind to T-cell immunoglobulin and mucin-domain containing-3 (TIM3) protein. Also provided are uses of these antibodies, or antigen-binding portions thereof, in therapeutic applications, such as treatment of cancer. Further provided are cells that produce the antibodies, or antigen-binding portions thereof, polynucleotides encoding the heavy and/or light chain regions of the antibodies, or antigen-binding portions thereof, and vectors comprising the polynucleotides encoding the heavy and/or light chain regions of the antibodies, or antigen-binding portions thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/649,380, filed Jul. 13, 2017, which claims the benefit of U.S.Provisional Application Nos. 62/362,541, filed Jul. 14, 2016, and62/459,499, filed Feb. 15, 2017, each of which is hereby incorporated byreference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing in ASCIItext file (Name: 33338_0520003_SeqListing_ST25.txt; Size: 779,880 bytes;and Date of Creation: Jun. 12, 2018) filed with the application isherein incorporated by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

T-cell immunoglobulin and mucin-domain containing-3 (TIM3), also knownas hepatitis A virus cellular receptor 2 (HAVCR2), is a type-Itransmembrane protein that functions as a key regulator of immuneresponses. TIM3 was initially identified on activated IFN-γ producing Tcells (e.g., type 1 helper CD4⁺ T cells and cytotoxic CD8⁺ T cells) andshown to induce T cell death or exhaustion after binding to galectin-9.More recent studies have indicated that TIM3 expression is alsoimportant in regulating the activities of many innate immune cells(e.g., macrophages, monocytes, dendritic cells, mast cells, and naturalkiller cells). See Han G et al., Front Immunol. 4: 449 (2013).

Like many inhibitory receptors (e.g., PD-1 and CTLA-4), TIM3 expressionhas been associated with many types of chronic diseases, includingcancer. TIM3⁺ T cells have been detected in patients with advancedmelanoma, non-small cell lung cancer, or follicular B-cell non-Hodgkinlymphoma. And the presence of TIM3⁺ regulatory T cells have beendescribed as an effective indicator of lung cancer progression. SeeAnderson A C. Cancer Immunol Res. 2: 393-8 (2014).

Several potential ligands for TIM3 have been identified: Galectin-9,HMGB 1, Semaphorin-4A, CEACAM-1, ILT-4 and phosphatidylserine (PtdSer orPS). PS is an important cell membrane component, and is normallylocalized to the inner leaflet of cell membranes. But as a cellundergoes apoptosis, PS is redistributed and exposed to the outermembrane. This redistribution is also observed in many tumor cell lines.See Riedl S et al., Blochim Blophys Acta. 1808: 2638-2645 (2011).Binding of TIM3 to PS may be critical for phagocytosis andcross-presentation. See Nakayama M et al., Blood. 113: 3821-30 (2009).

Studies have shown a close relationship between TIM3 and the inhibitoryreceptor PD-1. For example, many tumor-specific T cells express bothPD-1 and TIM3, and these T cells have been shown to be moredysfunctional compared to T cells that express only PD-1 or TIM3. SeeFourcade J et al., J Exp Med. 207: 2175-2186 (2010).

Accordingly, agents that target TIM3, and methods of using such agents,are highly desirable for designing new cancer immunotherapies andimproving traditional cancer immunotherapies.

SUMMARY OF THE DISCLOSURE

Provided herein are isolated antibodies, such as monoclonal antibodies,in particular human (e.g., monoclonal) antibodies, that specificallybind TIM3 and have desirable functional properties. These propertiesinclude, e.g., high affinity binding to human TIM3, binding to monkeyTIM3 (e.g., cynomolgus TIM3), and the ability to stimulate immuneresponses, e.g., antigen-specific T cell responses, such as in atumor-bearing or virus-bearing (virus-infected) subject, and to detectTIM3 protein in a sample.

In one aspect, the isolated antibodies, or antigen binding portionsthereof, which bind to TIM3, exhibit at least one of the followingproperties:

(a) binding to soluble and/or membrane bound human TIM3;

(b) binding to soluble and/or membrane bound cyno TIM3;

(c) inducing or stimulating an immune response;

(d) inducing or stimulating T cell activation, e.g., Th1 cellactivation, (as evidenced, e.g., by enhanced cytokine secretion and/orproliferation);

(e) inducing or stimulating T cell proliferation (e.g., CD4+, CD8+ Tcells, Th1 cells or TILs), e.g., in a coculture assay, such as describedin the Examples;

(f) inducing or stimulating IFN-γ production by T cells, e.g., Th1 cellsor tumor infiltrating lymphocytes (TILs), such as TILs from human renal,lung, pancreatic or breast cancer tumors, as determined, e.g., in theassay described in the Examples;

(g) blocking or inhibiting the binding of human TIM3 to PtdSer, asdetermined, e.g., in the assay described in the Examples;

(h) not internalizing or downregulating cell surface TIM3 when bindingto TIM3 on cells;

(i) binding to human TIM3 extracellular domain (a) CPVFECG (SEQ ID NO:296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQID NOs: 296 and 298, respectively); or (d) WTSRYWLNGDFR (SEQ ID NO:297);

(j) competing with, or cross-blocking, the binding to human TIM3 of anantibody binding to TIM3 described herein (e.g., 13A3, 3G4, 17C3, 17C8,9F6, or any of TIM3.2 to TIM3.18), as determined, e.g., in the assaydescribed in the Examples;

(k) binding to human TIM3, but not to human TIM3 having an amino acidsubstitution of one or more of the following amino acid residues: L48,C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87,R89, D104, R111, Q113, G116, M118, and D120, as numbered in SEQ ID NO:286 (FIG. 20);(l) binding to human TIM3 regions ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367),¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368), and ¹¹⁹NDEKFNLKL¹²⁷ (SEQ IDNO: 373) as determined by HDX-MS;(m) having the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography; and/or(n) competing with or cross-blocking with the binding to human TIM3 of13A3 or TIM3.18.IgG1.3, e.g., as described in the Examples.

In certain embodiments, the anti-TIM3 antibodies, or antigen bindingportion thereof, stimulate an anti-tumor immune response, e.g., anantigen-specific T cell response. In other embodiments, the anti-TIM3antibodies, or antigen binding portions thereof, increase cytokineproduction (e.g., IFN-γ) in TIM3-expressing T cells and/or increase Tcell proliferation. In some embodiments, the anti-TIM3 antibodies, orantigen binding portions thereof, do not bind to Fc receptors.

In certain embodiments, the anti-TIM3 antibodies, or antigen bindingportions thereof, bind to soluble human TIM3 with a K_(D) of 10 nM orless as measured by Biacore, bind to membrane bound human TIM3 with aK_(D) of 1 nM or less as measured by Scatchard, bind to solublecynomolgus TIM3 with a K_(D) of 100 nM or less as measured by Biacore,bind to membrane bound human TIM3 with an EC₅₀ of 1 μg/mL or less asmeasured by flow cytometry, bind to membrane bound human TIM3 with anEC₅₀ of 0.1 μg/mL or less as measured by flow cytometry, bind tomembrane bound cynomolgus TIM3 with an EC₅₀ of 1 μg/mL or less asmeasured by flow cytometry, bind to membrane bound cyno TIM-3 with aK_(D) of 1 nM or less as measured by Scatchard.

Provided herein are isolated antibodies, or antigen binding portionsthereof, which bind to human TIM3 and comprise heavy chain CDR1, CDR2,and CDR3 and light chain CDR1, CDR2, and CDR3, wherein the heavy chainCDR3 comprises an amino acid sequence selected from the group consistingof: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ IDNO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 126, SEQ ID NO: 127,SEQ ID NO: 128, and SEQ ID NO: 129.

In certain embodiments, the heavy chain CDR1 comprises X1, X2, X3, X4,Y, X5, and X6, and wherein X1 is S or none, X2 is R or none, X3 is S, R,or D, X4 is Y or H, X5 is W or M, and X6 is G, N, S, or H. In otherembodiments, the heavy chain CDR1 comprises X1, Y, Y, M, and X2, andwherein X1 is S or D and X2 is H or S. In some embodiments, the heavychain CDR1 comprises R, X1, Y, W, and X2, and wherein X1 is H or Y andX2 is N or S.

In one embodiment, the heavy chain CDR2 comprises X1, I, X2, X3, X4, G,X5, X6, X7, X8, Y, X9, X10, X11, X12, X13, and X14, and wherein X1 is S,Y, I, or F, X2 is Y, H, N, or S, X3 is Y, P, G, T, or S, X4 is S, T, R,or G, X5 is F, S, or D, X6 is S, T, or I, X7 is I or none, X8 is Y, N,or I, X9 is N, Q, S, or A, X10 is P, S, Q, or D, X11 is S or K, X12 isL, F, or V, X13 is K or Q, and X14 is S or G. In another embodiment, theheavy chain CDR2 comprises Y, I, H, Y, X1, G, S, T, N, Y, N, X2, S, L,K, and S, and wherein X1 is S or T and X2 is S or P. In someembodiments, the heavy chain CDR2 comprises F, I, S, X1, X2, G, S, X3,I, Y, Y, A, D, S, V, K, and G, and wherein X1 is G, T or S, X2 is G orS, and X3 is T or I. In other embodiments, the heavy chain CDR2comprises I, I, N, P, R, G, D, S, I, I, Y, A, Q, K, F, Q, and G.

In certain embodiments, the anti-TIM3 antibodies, or antigen bindingportions thereof, comprise a light chain CDR1 comprising SEQ ID NO: 64or SEQ ID NO: 65, a light chain CDR2 comprising SEQ ID NO: 66 or SEQ IDNO: 67, and/or a light chain CDR3 comprising SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO: 70, or SEQ ID NO: 71.

Provided herein are isolated antibodies, or antigen binding portionsthereof, which bind human TIM3 and comprise heavy chain CDR1, CDR2, andCDR3 and light chain CDR1, CDR2, and CDR3, wherein

(a) the heavy chain CDR1 is selected from the group consisting of SEQ IDNO: 41, SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; and SEQ ID NO: 45;

(b) the heavy chain CDR2 is selected from the group consisting of SEQ IDNO: 46, SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQID NO: 51; SEQ ID NO: 52; SEQ ID NO: 122; SEQ ID NO: 123; SEQ ID NO: 124and SEQ ID NO: 125;

(c) the heavy chain CDR3 is selected from the group consisting of SEQ IDNO: 53, SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQID NO: 58; SEQ ID NO: 59; SEQ ID NO: 126; SEQ ID NO: 127; SEQ ID NO: 128and SEQ ID NO: 129;

(d) the light chain CDR1 comprises SEQ ID NO: 64 or SEQ ID NO: 65;

(e) the light chain CDR2 comprises SEQ ID NO: 66 or SEQ ID NO: 67; and

(f) the light chain CDR3 comprises SEQ ID NO: 68, SEQ ID NO: 69, SEQ IDNO: 70, or SEQ ID NO: 71.

Provided herein are isolated antibodies, or antigen binding portionsthereof, which bind to human TIM3 and comprise:

(a1) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a2) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 122, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a3) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 123, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a4) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 124, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a5) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 126, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a6) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 127, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a7) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 128, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a8) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 129, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a9) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 122, 128, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a10) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 122, 126, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(b1) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 42, 47, 54, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;

(b2) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 42, 125, 54, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:43, 48, and 55, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 69, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:44, 49, and 56, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 68, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:45, 50, and 57, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 69, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:45, 50, and 57, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 71, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:45, 50, and 57, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 65, 67, and 70, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:45, 51, and 58, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 68, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:45, 52, and 59, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 69, respectively.

Provided herein are isolated antibodies, or antigen binding portionsthereof, which bind to human TIM3 and comprise heavy and light chainvariable regions, wherein the heavy chain variable region comprises anamino acid sequence which is at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, or about 100% identical tothe amino acid sequence selected from the group consisting of SEQ IDNOs: 34, 35, 36, 37, 38, 39, 40, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, and 364 and/or wherein the light chain variable regioncomprises an amino acid sequence which is at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or about100% identical to the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 60, 61, 62, and 63.

Provided herein are isolated antibodies, or antigen binding portionsthereof, which bind to human TIM3 and cross-compete for binding to humanTIM3 with a reference antibody comprising a VH and a VL, wherein the VHand the VL are selected from the group consisting of:

(a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 34and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60;

(b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 35and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61;

(c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 36and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61;

(d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 37and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60;

(e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61;

(f) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38and a VL comprising the amino acid sequence set forth in SEQ ID NO: 62;

(g) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38and a VL comprising the amino acid sequence set forth in SEQ ID NO: 63;

(h) a VH comprising the amino acid sequence set forth in SEQ ID NO: 39and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60;

(i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 40and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61;

(j) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 121and a VL comprising the amino acid sequence set forth in 63,respectively;

(k) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 120and a VL comprising the amino acid sequence set forth in 61,respectively;

(l) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 112and a VL comprising the amino acid sequence set forth in 60,respectively;

(m) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 113and a VL comprising the amino acid sequence set forth in 60,respectively;

(n) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 114and a VL comprising the amino acid sequence set forth in 60,respectively;

(o) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 115and a VL comprising the amino acid sequence set forth in 60,respectively;

(p) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 116and a VL comprising the amino acid sequence set forth in 60,respectively;

(q) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 117and a VL comprising the amino acid sequence set forth in 60,respectively;

(r) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 118and a VL comprising the amino acid sequence set forth in 60,respectively;

(s) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 119and a VL comprising the amino acid sequence set forth in 60,respectively; and

(t) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 364and a VL comprising the amino acid sequence set forth in 60,respectively.

In one embodiment, the isolated anti-TIM3 antibodies, or antigen bindingportions thereof, bind to TIM3 at the same epitope as the referenceantibody.

In other embodiments, the isolated anti-TIM3 antibodies, or antigenbinding portions thereof, comprise a VH and a VL, selected from thegroup consisting of:

(a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 34and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60;

(b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 35and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61;

(c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 36and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61;

(d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 37and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60;

(e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61;

(f) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38and a VL comprising the amino acid sequence set forth in SEQ ID NO: 62;

(g) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38and a VL comprising the amino acid sequence set forth in SEQ ID NO: 63;

(h) a VH comprising the amino acid sequence set forth in SEQ ID NO: 39and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60;

(i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 40and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61;

(j) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 121and a VL comprising the amino acid sequence set forth in 63,respectively;

(k) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 120and a VL comprising the amino acid sequence set forth in 61,respectively;

(l) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 112and a VL comprising the amino acid sequence set forth in 60,respectively;

(m) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 113and a VL comprising the amino acid sequence set forth in 60,respectively;

(n) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 114and a VL comprising the amino acid sequence set forth in 60,respectively;

(o) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 115and a VL comprising the amino acid sequence set forth in 60,respectively;

(p) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 116and a VL comprising the amino acid sequence set forth in 60,respectively;

(q) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 117and a VL comprising the amino acid sequence set forth in 60,respectively;

(r) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 118and a VL comprising the amino acid sequence set forth in 60,respectively;

(s) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 119and a VL comprising the amino acid sequence set forth in 60,respectively; and

(t) a VH comprising the amino acid sequence set forth in SEQ ID NOs: 364and a VL comprising the amino acid sequence set forth in 60,respectively.

In certain embodiments, the anti-TIM3 antibodies, or antigen bindingportions thereof, are selected from the group consisting of an IgG1, anIgG2, an IgG3, an IgG4 or a variant thereof. In some embodiments, theanti-TIM3 antibodies, or antigen binding portions thereof, comprise aneffectorless IgG1 Fc that comprises the following mutations: L234A,L235E, G237A, and optionally A330S and P331S. In other embodiments, theanti-TIM3 antibodies, or antigen binding portions thereof, comprise aheavy chain constant region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 130-133. In certainembodiments, the anti-TIM3 antibodies, or antigen binding portionsthereof, are human or humanized antibody.

In certain embodiments, the anti-TIM3 antibodies, or antigen bindingportions thereof, specifically bind to human TIM3 and comprise

(a1) heavy and light chain sequences comprising SEQ ID NOs: 301 (or 302)and 29, respectively;

(a2) heavy and light chain sequences comprising SEQ ID NOs: 1 (or 8) and29, respectively;

(a3) heavy and light chain sequences comprising SEQ ID NOs: 15 (or 22)and 29, respectively;

(a4) heavy and light chain sequences comprising SEQ ID NOs: 303 (or 304)and 29, respectively;

(a5) heavy and light chain sequences comprising SEQ ID NOs: 72 (or 82)and 29, respectively;

(a6) heavy and light chain sequences comprising SEQ ID NOs: 92 (or 102)and 29, respectively;

(a7) heavy and light chain sequences comprising SEQ ID NOs: 305 (or 306)and 29, respectively;

(a8) heavy and light chain sequences comprising SEQ ID NOs: 73 (or 83)and 29, respectively;

(a9) heavy and light chain sequences comprising SEQ ID NOs: 93 (or 103)and 29, respectively;

(a10) heavy and light chain sequences comprising SEQ ID NOs: 307 (or308) and 29, respectively;

(a11) heavy and light chain sequences comprising SEQ ID NOs: 74 (or 84)and 29, respectively;

(a12) heavy and light chain sequences comprising SEQ ID NOs: 94 (or 104)and 29, respectively;

(a13) heavy and light chain sequences comprising SEQ ID NOs: 309 (or310) and 29, respectively;

(a14) heavy and light chain sequences comprising SEQ ID NOs: 75 (or 85)and 29, respectively;

(a15) heavy and light chain sequences comprising SEQ ID NOs: 95 (or 105)and 29, respectively;

(a16) heavy and light chain sequences comprising SEQ ID NOs: 311 (or312) and 29, respectively;

(a17) heavy and light chain sequences comprising SEQ ID NOs: 76 (or 86)and 29, respectively;

(a18) heavy and light chain sequences comprising SEQ ID NOs: 96 (or 106)and 29, respectively;

(a19) heavy and light chain sequences comprising SEQ ID NOs: 313 (or314) and 29, respectively;

(a20) heavy and light chain sequences comprising SEQ ID NOs: 77 (or 87)and 29, respectively;

(a21) heavy and light chain sequences comprising SEQ ID NOs: 97 (or 107)and 29, respectively;

(a22) heavy and light chain sequences comprising SEQ ID NOs: 315 (or316) and 29, respectively;

(a23) heavy and light chain sequences comprising SEQ ID NOs: 78 (or 88)and 29, respectively;

(a24) heavy and light chain sequences comprising SEQ ID NOs: 98 (or 108)and 29, respectively;

(a25) heavy and light chain sequences comprising SEQ ID NOs: 317 (or318) and 29, respectively;

(a26) heavy and light chain sequences comprising SEQ ID NOs: 79 (or 89)and 29, respectively;

(a27) heavy and light chain sequences comprising SEQ ID NOs: 99 (or 109)and 29, respectively;

(a28) heavy and light chain sequences comprising SEQ ID NOs: 319 (or320) and 29, respectively;

(a29) heavy and light chain sequences comprising SEQ ID NOs: 349 (or350) and 29, respectively;

(a30) heavy and light chain sequences comprising SEQ ID NOs: 351 (or352) and 29, respectively;

(a31) heavy and light chain sequences comprising SEQ ID NOs: 353 (or354) and 29, respectively;

(b1) heavy and light chain sequences comprising SEQ ID NOs: 321 (or 322)and 30, respectively;

(b2) heavy and light chain sequences comprising SEQ ID NOs: 2 (or 9) and30, respectively;

(b3) heavy and light chain sequences comprising SEQ ID NOs: 16 (or 23)and 30, respectively;

(b4) heavy and light chain sequences comprising SEQ ID NOs: 323 (or 324)and 30, respectively;

(b5) heavy and light chain sequences comprising SEQ ID NOs: 80 (or 90)and 30, respectively;

(b6) heavy and light chain sequences comprising SEQ ID NOs: 100 (or 110)and 30, respectively;

(b7) heavy and light chain sequences comprising SEQ ID NOs: 325 (or 326)and 30, respectively;

(c1) heavy and light chain sequences comprising SEQ ID NOs: 327 (or 328)and 30, respectively;

(c2) heavy and light chain sequences comprising SEQ ID NOs: 3 (or 10)and 30, respectively;

(c3) heavy and light chain sequences comprising SEQ ID NOs: 17 (or 24)and 30, respectively;

(c4) heavy and light chain sequences comprising SEQ ID NOs: 329 (or 330)and 30, respectively;

(d1) heavy and light chain sequences comprising SEQ ID NOs: 331 (or 332)and 29, respectively;

(d2) heavy and light chain sequences comprising SEQ ID NOs: 4 (or 11)and 29, respectively;

(d3) heavy and light chain sequences comprising SEQ ID NOs: 18 (or 25)and 29, respectively;

(d4) heavy and light chain sequences comprising SEQ ID NOs: 333 (or 334)and 29, respectively;

(e1) heavy and light chain sequences comprising SEQ ID NOs: 335 (or 336)and 32, respectively;

(e12) heavy and light chain sequences comprising SEQ ID NOs: 335 (or336) and 33, respectively;

(e13) heavy and light chain sequences comprising SEQ ID NOs: 335 (or336) and 33, respectively;

(e2) heavy and light chain sequences comprising SEQ ID NOs: 5 (or 12)and 33, respectively;

(e3) heavy and light chain sequences comprising SEQ ID NOs: 19 (or 26)and 33, respectively;

(e4) heavy and light chain sequences comprising SEQ ID NOs: 337 (or 338)and 33, respectively;

(e5) heavy and light chain sequences comprising SEQ ID NOs: 81 (or 91)and 33, respectively;

(e6) heavy and light chain sequences comprising SEQ ID NOs: 101 (or 111)and 33, respectively;

(e7) heavy and light chain sequences comprising SEQ ID NOs: 339 (or 340)and 33, respectively;

(f1) heavy and light chain sequences comprising SEQ ID NOs: 341 (or 342)and 29, respectively;

(f2) heavy and light chain sequences comprising SEQ ID NOs: 6 (or 13)and 29, respectively;

(f3) heavy and light chain sequences comprising SEQ ID NOs: 20 (or 27)and 29, respectively;

(f4) heavy and light chain sequences comprising SEQ ID NOs: 343 (or 344)and 29, respectively;

(g1) heavy and light chain sequences comprising SEQ ID NOs: 345 (or 346)and 30, respectively;

(g2) heavy and light chain sequences comprising SEQ ID NOs: 7 (or 14)and 30, respectively;

(g3) heavy and light chain sequences comprising SEQ ID NOs: 21 (or 28)and 30, respectively; or

(g4) heavy and light chain sequences comprising SEQ ID NOs: 347 (or 348)and 30, respectively.

In other embodiments, the anti-TIM3 antibodies, or antigen bindingportions thereof, have one or more of the following properties:

(1) binding to soluble human TIM3, e.g., with a K_(D) of 10 nM or less(e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;

(2) binding to soluble cynomolgus TIM3, e.g., with a K_(D) of 100 nM orless (e.g., 0.01 nM to 100 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;

(3) binding to membrane bound human TIM3, e.g., with an EC₅₀ of 1 ug/mLor less (e.g., 0.01 ug/mL to 1 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);

(4) binding to membrane bound human TIM3, e.g., with a K_(D) of 1 nM orless (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard analysis,e.g., as described in the Examples;

(5) binding to membrane bound cynomolgus TIM3, e.g., with an EC₅₀ of 20ug/mL or less (e.g., 0.01 ug/mL to 20 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);

(6) binding to membrane bound cynomolgus TIM3, e.g., with a K_(D) of 1nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchardanalysis, e.g., as described in the Examples;

(7) inducing or enhancing T cell activation (e.g., by blocking orreducing the inhibitory effect of TIM3), as evidenced by (i) increasedIFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs)and/or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1cells or TILs), e.g., as described in the Examples;(8) stimulating T cell proliferation in a mixed lymphocyte reaction(MLR) assay, e.g., as described in the Examples;(9) inhibiting the binding of phosphatidylserine to TIM3, e.g., asmeasured by PS-hTIM3 “in-tandem” blocking assay, e.g., as described inthe Examples;(10) not internalizing or downregulating cell surface TIM3 when bindingto TIM3 on cells;(11) binding to one of the following regions of human TIM3 extracellulardomain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND(SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298,respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297), e.g., as describedin the Examples;(12) having reduced binding to human TIM3 in which one or more of aminoacids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84,G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (as numbered inSEQ ID NO: 286 (FIG. 20)) is substituted with another amino acidrelative to binding to wild-type human TIM3, e.g., as described in theExamples;(13) competing in either direction or both directions for binding tohuman TIM3 with an antibody comprising VH and VL domains of any one of13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or TIM3.7, TIM3.8, TIM3.10,TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, andTIM3.18, e.g., as described in the Examples;(14) binding to human TIM3 regions ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367)and ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) as determined by HDX-MS,e.g., as described in the Examples;(15) having the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography (e.g., describedin the Examples; numbering per SEQ ID NO: 286 (FIG. 20)); and/or(16) (a) having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6,7, 8 or 9 of amino acids C58, P59, F61, E62, C63, R111, D120, andoptionally D104 and Q113 (numbering per SEQ ID NO: 286 (FIG. 20)) aresubstituted with another amino acid relative to binding to wildtypehuman TIM3; (b) binding to ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367),¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368), and ¹¹⁹NDEKFNLKL¹²⁷ (SEQ IDNO: 373), as determined by HDX-MS, as described in the Examples; and/or(c) competing with or cross-blocking with the binding to human TIM3 of13A3 or TIM3.18.IgG1.3, e.g., as described in the Examples.

Provided herein are bispecific molecules comprising an anti-TIM3antibody linked to a molecule having a second binding specificity.

Provided herein are nucleic acids encoding the heavy and/or light chainvariable regions of the anti-TIM3 antibodies, or antigen bindingportions thereof, expression vectors comprising the nucleic acidmolecules, and cells transformed with the expression vectors.

Provided herein are immunoconjugates comprising the anti-TIM3 antibodiesdescribed herein, linked to an agent.

Provided herein are compositions comprising anti-TIM3 antibodies, orantigen binding portions thereof, bispecific molecules, orimmunoconjugates described herein, and a carrier. Also provided hereinare kits comprising the anti-TIM3 antibodies, or antigen bindingportions thereof, bispecific molecules, or immunoconjugates describedherein, and instructions for use.

Provided herein is a method of preparing anti-TIM3 antibodies, orantigen binding portions thereof, comprising expressing an anti-TIM3antibody, or antigen binding portion thereof, in a cell and isolatingthe antibody, or antigen binding portion thereof, from the cell.

Provided herein is a method of stimulating an antigen-specific T cellresponse comprising contacting the T cell with an anti-TIM3 antibody, orantigen binding portion thereof, bispecific molecules, orimmunoconjugates described herein such that an antigen-specific T cellresponse is stimulated (e.g., by inhibiting the negative effect of TIM3on cells, e.g., T cells).

Provided herein is a method of activating or co-stimulating a T cell,e.g., an effector T cell (e.g., Th1 cell), comprising contacting a cell,e.g., an effector T cell, with an anti-TIM3 antibody, or antigen bindingportion thereof, bispecific molecules, or immunoconjugates describedherein, and CD3, wherein the effector T cell is activated orco-stimulated (e.g., by inhibiting the negative effect of TIM3 on cells,e.g., T cells).

Provided herein is a method of increasing IFN-γ production in and/orproliferation of a T cell, e.g., Th1 cell or TIL, comprising contactingthe T cell with an effective amount of an anti-TIM3 antibody, or antigenbinding portion thereof, bispecific molecules, or immunoconjugatesdescribed herein.

Provided herein is a method of increasing IFN-γ production in T cells ina subject comprising administering to the subject an effective amount ofan anti-TIM3 antibody, or antigen binding portion thereof, bispecificmolecule, or immunoconjugate described herein to increase IFN-γproduction from the T cells.

Provided herein a method of stimulating TIL activity in a subjectcomprising administering to the subject a therapeutically effectiveamount of an anti-TIM3 antibody, or antigen binding portion thereof,described herein, such that the TILs proliferate or secrete a cytokine,e.g., IFN-γ.

Provided herein are methods for stimulating NK cells (e.g., byincreasing NK cell cytotoxic activity) and/or macrophages or otherantigen presenting cell in a subject, comprising administering to thesubject an effective amount of an anti-TIM3 antibody, or antigen bindingportion thereof, bispecific molecule, or immunoconjugate describedherein. For example, an anti-TIM3 antibody described herein can increaseIL-12 secretion by antigen presenting cells contacted with the TIM3antibody.

Provided herein is a method of stimulating an immune response in asubject comprising administering to the subject an anti-TIM3 antibody,or antigen binding portion thereof, bispecific molecule orimmunoconjugate described herein, such that an immune response in thesubject is stimulated. In certain embodiments, the subject has a tumorand an immune response against the tumor is stimulated.

Provided herein is a method for inhibiting the growth of tumors orreducing the size of tumors in a subject comprising administering to thesubject an anti-TIM3 antibody, or antigen binding portion thereof,bispecific molecule, or immunoconjugate described herein, such thatgrowth of the tumor is inhibited in the subject.

Provided herein is a method of treating cancer, e.g., by immunotherapy,comprising administering to a subject in need thereof a therapeuticallyeffective amount of an anti-TIM3 antibody, or antigen binding portionthereof, bispecific molecule, or immunoconjugate described herein totreat the cancer. In certain embodiments, the cancer is selected fromthe group consisting of: bladder cancer, breast cancer, uterine/cervicalcancer, ovarian cancer, prostate cancer, testicular cancer, esophagealcancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer,colon cancer, kidney cancer, head and neck cancer, lung cancer, stomachcancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer,skin cancer, neoplasm of the central nervous system, lymphoma, leukemia,myeloma, sarcoma, virus-related cancer, and any combinations thereof. Insome embodiments, the cancer is a metastatic cancer, refractory cancer,or recurrent cancer. In some embodiments, the cancer is a cold tumor.

In certain embodiments, the methods described herein further compriseone or more additional therapeutics with an anti-TIM3 antibody, e.g., ananti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, ananti-GITR antibody, and/or an anti-PD-L1 antibody.

Provided herein is a method of detecting the presence of a TIM3 proteinin a sample comprising contacting the sample with an anti-TIM3 antibody,or antigen binding portion thereof, under conditions that allow forformation of a complex between the antibody, or antigen binding portionthereof, and TIM3, and detecting the formation of a complex.

EMBODIMENTS Embodiment 1

An isolated antibody (e.g., a human antibody), or antigen bindingportion thereof, which binds to human T-cell immunoglobulin andmucin-domain containing-3 (TIM3), wherein the antibody or antigenbinding portion thereof comprises heavy chain CDR1, CDR2, and CDR3 andlight chain CDR1, CDR2, and CDR3, wherein

(a) the heavy chain CDR1 is selected from the group consisting of SEQ IDNO: 41, SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; and SEQ ID NO: 45;

(b) the heavy chain CDR2 is selected from the group consisting of SEQ IDNO: 46, SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQID NO: 51; SEQ ID NO: 52; SEQ ID NO: 122; SEQ ID NO: 123; SEQ ID NO: 124and SEQ ID NO: 125;

(c) the heavy chain CDR3 is selected from the group consisting of SEQ IDNO: 53, SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQID NO: 58; SEQ ID NO: 59; SEQ ID NO: 126; SEQ ID NO: 127; SEQ ID NO: 128and SEQ ID NO: 129;

(d) the light chain CDR1 comprises SEQ ID NO: 64 or SEQ ID NO: 65;

(e) the light chain CDR2 comprises SEQ ID NO: 66 or SEQ ID NO: 67; and

(f) the light chain CDR3 comprises SEQ ID NO: 68, SEQ ID NO: 69, SEQ IDNO: 70, or SEQ ID NO: 71.

Embodiment 2

An isolated antibody, or antigen binding portion thereof, which binds tohuman TIM3, comprising:

(a1) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a2) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 122, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a3) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 123, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a4) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 124, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a5) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 126, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a6) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 127, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a7) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 128, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a8) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 129, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a9) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 122, 128, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a10) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 122, 126, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(b1) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 42, 47, 54, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;

(b2) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 42, 125, 54, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:43, 48, and 55, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 69, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:44, 49, and 56, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 68, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:45, 50, and 57, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 69, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:45, 50, and 57, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 71, respectively;

(g1) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:45, 50, and 57, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 65, 67, and 70, respectively;

(g2) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 45, 50, 57, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 71, respectively;

(g3) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 45, 50, 57, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:45, 51, and 58, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 68, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:45, 52, and 59, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 64, 66, and 69, respectively.

Embodiment 3

The antibody, or antigen binding portion thereof, of Embodiment 1 or 2,wherein the heavy chain variable region comprises an amino acid sequencewhich is at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or about 100% identical to the amino acidsequence selected from the group consisting of SEQ ID NOs: 34, 35, 36,37, 38, 39, 40, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and364 and/or the light chain variable region comprises an amino acidsequence which is at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or about 100% identical to theamino acid sequence selected from the group consisting of SEQ ID NOs:60, 61, 62, and 63.

Embodiment 4

The antibody, or antigen binding portion thereof, of any one ofEmbodiments 1 to 3, wherein the antibody, or antigen binding portionthereof, comprises an effectorless IgG1 Fc that comprises the followingmutations: L234A, L235E, G237A, and optionally A330S and P331S.

Embodiment 5

The antibody, or antigen binding portion thereof, of any of thepreceding Embodiments, comprising a heavy chain constant regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 263-266.

Embodiment 6

The antibody, or antigen binding portion thereof, of any of thepreceding Embodiments, wherein the antibody, or antigen binding portionthereof, is a human or humanized antibody.

Embodiment 7

The antibody of any one of Embodiments 1-6, wherein the antibodycomprises:

(a1) heavy and light chain sequences comprising SEQ ID NOs: 301 (or 302)and 29, respectively;

(a2) heavy and light chain sequences comprising SEQ ID NOs: 1 (or 8) and29, respectively;

(a3) heavy and light chain sequences comprising SEQ ID NOs: 15 (or 22)and 29, respectively;

(a4) heavy and light chain sequences comprising SEQ ID NOs: 303 (or 304)and 29, respectively;

(a5) heavy and light chain sequences comprising SEQ ID NOs: 72 (or 82)and 29, respectively;

(a6) heavy and light chain sequences comprising SEQ ID NOs: 92 (or 102)and 29, respectively;

(a7) heavy and light chain sequences comprising SEQ ID NOs: 305 (or 306)and 29, respectively;

(a8) heavy and light chain sequences comprising SEQ ID NOs: 73 (or 83)and 29, respectively;

(a9) heavy and light chain sequences comprising SEQ ID NOs: 93 (or 103)and 29, respectively;

(a10) heavy and light chain sequences comprising SEQ ID NOs: 307 (or308) and 29, respectively;

(a11) heavy and light chain sequences comprising SEQ ID NOs: 74 (or 84)and 29, respectively;

(a12) heavy and light chain sequences comprising SEQ ID NOs: 94 (or 104)and 29, respectively;

(a13) heavy and light chain sequences comprising SEQ ID NOs: 309 (or310) and 29, respectively;

(a14) heavy and light chain sequences comprising SEQ ID NOs: 75 (or 85)and 29, respectively;

(a15) heavy and light chain sequences comprising SEQ ID NOs: 95 (or 105)and 29, respectively;

(a16) heavy and light chain sequences comprising SEQ ID NOs: 311 (or312) and 29, respectively;

(a17) heavy and light chain sequences comprising SEQ ID NOs: 76 (or 86)and 29, respectively;

(a18) heavy and light chain sequences comprising SEQ ID NOs: 96 (or 106)and 29, respectively;

(a19) heavy and light chain sequences comprising SEQ ID NOs: 313 (or314) and 29, respectively;

(a20) heavy and light chain sequences comprising SEQ ID NOs: 77 (or 87)and 29, respectively;

(a21) heavy and light chain sequences comprising SEQ ID NOs: 97 (or 107)and 29, respectively;

(a22) heavy and light chain sequences comprising SEQ ID NOs: 315 (or316) and 29, respectively;

(a23) heavy and light chain sequences comprising SEQ ID NOs: 78 (or 88)and 29, respectively;

(a24) heavy and light chain sequences comprising SEQ ID NOs: 98 (or 108)and 29, respectively;

(a25) heavy and light chain sequences comprising SEQ ID NOs: 317 (or318) and 29, respectively;

(a26) heavy and light chain sequences comprising SEQ ID NOs: 79 (or 89)and 29, respectively;

(a27) heavy and light chain sequences comprising SEQ ID NOs: 99 (or 109)and 29, respectively;

(a28) heavy and light chain sequences comprising SEQ ID NOs: 319 (or320) and 29, respectively;

(a29) heavy and light chain sequences comprising SEQ ID NOs: 349 (or350) and 29, respectively;

(a30) heavy and light chain sequences comprising SEQ ID NOs: 351 (or352) and 29, respectively;

(a31) heavy and light chain sequences comprising SEQ ID NOs: 353 (or354) and 29, respectively;

(b1) heavy and light chain sequences comprising SEQ ID NOs: 321 (or 322)and 30, respectively;

(b2) heavy and light chain sequences comprising SEQ ID NOs: 2 (or 9) and30, respectively;

(b3) heavy and light chain sequences comprising SEQ ID NOs: 16 (or 23)and 30, respectively;

(b4) heavy and light chain sequences comprising SEQ ID NOs: 323 (or 324)and 30, respectively;

(b5) heavy and light chain sequences comprising SEQ ID NOs: 80 (or 90)and 30, respectively;

(b6) heavy and light chain sequences comprising SEQ ID NOs: 100 (or 110)and 30, respectively;

(b7) heavy and light chain sequences comprising SEQ ID NOs: 325 (or 326)and 30, respectively;

(c1) heavy and light chain sequences comprising SEQ ID NOs: 327 (or 328)and 30, respectively;

(c2) heavy and light chain sequences comprising SEQ ID NOs: 3 (or 10)and 30, respectively;

(c3) heavy and light chain sequences comprising SEQ ID NOs: 17 (or 24)and 30, respectively;

(c4) heavy and light chain sequences comprising SEQ ID NOs: 329 (or 330)and 30, respectively;

(d1) heavy and light chain sequences comprising SEQ ID NOs: 331 (or 332)and 29, respectively;

(d2) heavy and light chain sequences comprising SEQ ID NOs: 4 (or 11)and 29, respectively;

(d3) heavy and light chain sequences comprising SEQ ID NOs: 18 (or 25)and 29, respectively;

(d4) heavy and light chain sequences comprising SEQ ID NOs: 333 (or 334)and 29, respectively;

(e1.1) heavy and light chain sequences comprising SEQ ID NOs: 335 (or336) and 32, respectively;

(e1.2) heavy and light chain sequences comprising SEQ ID NOs: 335 (or336) and 33, respectively;

(e1.3) heavy and light chain sequences comprising SEQ ID NOs: 335 (or336) and 31, respectively;

(e2) heavy and light chain sequences comprising SEQ ID NOs: 5 (or 12)and 33, respectively;

(e3) heavy and light chain sequences comprising SEQ ID NOs: 19 (or 26)and 33, respectively;

(e4) heavy and light chain sequences comprising SEQ ID NOs: 337 (or 338)and 33, respectively;

(e5) heavy and light chain sequences comprising SEQ ID NOs: 81 (or 91)and 33, respectively;

(e6) heavy and light chain sequences comprising SEQ ID NOs: 101 (or 111)and 33, respectively;

(e7) heavy and light chain sequences comprising SEQ ID NOs: 339 (or 340)and 33, respectively;

(f1) heavy and light chain sequences comprising SEQ ID NOs: 341 (or 342)and 29, respectively;

(f2) heavy and light chain sequences comprising SEQ ID NOs: 6 (or 13)and 29, respectively;

(f3) heavy and light chain sequences comprising SEQ ID NOs: 20 (or 27)and 29, respectively;

(f4) heavy and light chain sequences comprising SEQ ID NOs: 343 (or 344)and 29, respectively;

(g1) heavy and light chain sequences comprising SEQ ID NOs: 345 (or 346)and 29, respectively;

(g2) heavy and light chain sequences comprising SEQ ID NOs: 7 (or 43)and 30, respectively;

(g3) heavy and light chain sequences comprising SEQ ID NOs: 21 (or 28)and 30, respectively; or

(g4) heavy and light chain sequences comprising SEQ ID NOs: 347 (or 348)and 30, respectively; wherein the antibody specifically binds to humanTIM3.

Embodiment 8

The antibody or antigen binding portion thereof, of any of Embodiments1-7, wherein the antibody or antigen binding portion thereof has one ormore of the following properties:

(1) binding to soluble human TIM3, e.g., with a KD of 10 nM or less(e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;

(2) binding to soluble cynomolgus TIM3, e.g., with a KD of 100 nM orless (e.g., 0.01 nM to 100 nM), e.g., as measured by Biacore;

(3) binding to membrane bound human TIM3, e.g., with an EC50 of 1 ug/mLor less (e.g., 0.01 ug/mL to 1 ug/mL), e.g., as measured by flowcytometry;

(4) binding to membrane bound human TIM3, e.g., with a KD of 1 nM orless (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard analysis;

(5) binding to membrane bound cynomolgus TIM3, e.g., with an EC50 of 20ug/mL or less (e.g., 0.01 ug/mL to 20 ug/mL), e.g., as measured by flowcytometry;

(6) binding to membrane bound cynomolgus TIM3, e.g., with a KD of 1 nMor less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchardanalysis;

(7) inducing or enhancing T cell activation (e.g., by blocking orreducing the inhibitory effect of TIM3), as evidenced by (i) increasedIFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs)and/or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1cells or TILs);(8) stimulating T cell proliferation in a mixed lymphocyte reaction(MLR) assay;(9) inhibiting the binding of phosphatidylserine to TIM3, e.g., asmeasured by PS-hTIM3 “in-tandem” blocking assay;(10) not internalizing or downregulating cell surface TIM3 when bindingto TIM3 on cells;(11) binding to one of the following regions of human TIM3 extracellulardomain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND(SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298,respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297);(12) having reduced binding to human TIM3 in which one or more of aminoacids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84,G86, D87, R89, D104, R111, Q113, G116, M118, and D120 is substitutedwith another amino acid relative to binding to wildtype human TIM3;(13) competing in either direction or both directions for binding tohuman TIM3 with an antibody comprising VH and VL domains of any one of13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or TIM3.7, TIM3.8, TIM3.10,TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, andTIM3.18;(14) binding to human TIM3 regions 49VPVCWGKGACPVFE62 (SEQ ID NO: 367)and ¹¹¹RIQIPGIMNDEKFNLKL127 (SEQ ID NO: 368) as determined by HDX-MS(15) having the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography (e.g., describedin the Examples; numbering per SEQ ID NO: 286 (FIG. 20)); and/or(16) (a) having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6,7, 8 or 9 of amino acids C58, P59, F61, E62, C63, R111, D120, andoptionally D104 and Q113 (numbering per SEQ ID NO: 286 (FIG. 20)) aresubstituted with another amino acid relative to binding to wildtypehuman TIM3; (b) binding to 49VPVCWGKGACPVFE62 (SEQ ID NO: 367),111RIQIPGIMNDEKFNLKL127 (SEQ ID NO: 368) and 119NDEKFNLKL127 (SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and/or (c)competing with or cross-blocking with the binding to human TIM3 of 13A3or TIM3.18.IgG1.3.

Embodiment 9

A bispecific molecule comprising the antibody of any one of thepreceding Embodiments linked to a molecule having a second bindingspecificity.

Embodiment 10

A nucleic acid encoding the heavy and/or light chain variable region ofthe antibody, or antigen binding portion thereof, of any one ofEmbodiments 1 to 8.

Embodiment 11

A cell transformed with the nucleic acid of Embodiment 10.

Embodiment 12

An immunoconjugate comprising the antibody according to any one ofEmbodiments 1 to 8, linked to an agent.

Embodiment 13

A composition comprising the antibody, or antigen binding portionthereof, bispecific molecule or immunoconjugate, of any one ofEmbodiments 1 to 9 and 12, and a carrier.

Embodiment 14

A kit comprising the antibody, or antigen binding portion thereof, orbispecific molecule, or immunoconjugate of any one of Embodiments 1 to 9and 12 and instructions for use.

Embodiment 15

A method of stimulating, increasing or modulating an immune response ina subject in need thereof or for treating cancer in a subject in needthereof, comprising administering the antibody, or antigen bindingportion thereof, bispecific molecule or immunoconjugate, of any one ofEmbodiments 1 to 9 and 12, wherein an antigen-specific T cell responseis stimulated, wherein the effector T cell is activated orco-stimulated, wherein IFN-γ production in a T cell is increased,wherein the number of T cells is increased, wherein TIL activity isstimulated, wherein the size of a tumor in the subject is reduced,wherein growth of a tumor in the subject is inhibited, or anycombination thereof, after the administration.

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples, which should beconstrued as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 167) and the aminoacid sequence (SEQ ID NO: 34) of the mature heavy chain variable (VH)region of the anti-TIM3 monoclonal antibody 13A3. The CDR1 (SEQ ID NO:41), CDR2 (SEQ ID NO: 46) and CDR3 (SEQ ID NO: 53) are delineated, andthe V, D and J germline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 193) and the aminoacid sequence (SEQ ID NO: 60) of the mature light chain variable (VL)region of the anti-TIM3 monoclonal antibody 13A3. The CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 68) are delineated, andthe V and J germline derivations are indicated.

FIG. 1C shows the nucleotide sequence (SEQ ID NO: 167) and the aminoacid sequence (SEQ ID NO: 34) of the heavy chain VH region of theanti-TIM3 monoclonal antibody 13A3 with a signal sequence (SEQ ID NOs:274 and 269, respectively), and the nucleotide sequence (SEQ ID NO: 193)and the amino acid sequence (SEQ ID NO: 60) of the light chain VL regionof the anti-TIM3 monoclonal antibody 13A3 with a signal sequence (SEQ IDNOs: 273 and 268, respectively).

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 168) and the aminoacid sequence (SEQ ID NO: 35) of the mature heavy chain variable (VH)region of the anti-TIM3 monoclonal antibody 8B9. The CDR1 (SEQ ID NO:42), CDR2 (SEQ ID NO: 47) and CDR3 (SEQ ID NO: 54) are delineated, andthe V, D and J germline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 194) and the aminoacid sequence (SEQ ID NO: 61) of the mature light chain variable (VL)region of the anti-TIM3 monoclonal antibody 8B9. The CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are delineated, andthe V and J germline derivations are indicated.

FIG. 2C shows the nucleotide sequence (SEQ ID NO: 168) and the aminoacid sequence (SEQ ID NO: 35) of the heavy chain VH region of theanti-TIM3 monoclonal antibody 8B9 with a signal sequence (SEQ ID NOs:274 and 269, respectively), and the nucleotide sequence (SEQ ID NO: 194)and the amino acid sequence (SEQ ID NO: 61) of the light chain VL regionof the anti-TIM3 monoclonal antibody 8B9 with a signal sequence (SEQ IDNOs: 0.273 and 268, respectively).

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 169) and the aminoacid sequence (SEQ ID NO: 36) of the mature heavy chain variable (VH)region of the anti-TIM3 monoclonal antibody 8C4. The CDR1 (SEQ ID NO:43), CDR2 (SEQ ID NO: 48) and CDR3 (SEQ ID NO: 55) are delineated, andthe V, D and J germline derivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 194) and the aminoacid sequence (SEQ ID NO: 61) of the mature light chain variable (VL)region of the anti-TIM3 monoclonal antibody 8C4. The CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are delineated, andthe V and J germline derivations are indicated.

FIG. 3C shows the nucleotide sequence (SEQ ID NO: 169) and the aminoacid sequence (SEQ ID NO: 36) of the heavy chain VH region of theanti-TIM3 monoclonal antibody 8C4 with a signal sequence (SEQ ID NOs:274 and 269, respectively), and the nucleotide sequence (SEQ ID NO: 194)and the amino acid sequence (SEQ ID NO: 61) of the light chain VL regionof the anti-TIM3 monoclonal antibody 8C4 with a signal sequence (SEQ IDNOs: 273 and 268, respectively).

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 170) and the aminoacid sequence (SEQ ID NO: 37) of the mature heavy chain variable (VH)region of the anti-TIM3 monoclonal antibody 17C3. The CDR1 (SEQ ID NO:44), CDR2 (SEQ ID NO: 49) and CDR3 (SEQ ID NO: 56) are delineated, andthe V, D and J germline derivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 193) and the aminoacid sequence (SEQ ID NO: 60) of the mature light chain variable (VL)region of the anti-TIM3 monoclonal antibody 17C3. The CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 68) are delineated, andthe V and J germline derivations are indicated.

FIG. 4C shows the nucleotide sequence (SEQ ID NO: 170) and the aminoacid sequence (SEQ ID NO: 37) of the heavy chain VH region of theanti-TIM3 monoclonal antibody 17C3 with a signal sequence (SEQ ID NOs:272 and 267, respectively), and the nucleotide sequence (SEQ ID NO: 193)and the amino acid sequence (SEQ ID NO: 60) of the light chain VL regionof the anti-TIM3 monoclonal antibody 17C3 with a signal sequence (SEQ IDNOs: 273 and 268, respectively).

FIG. 5A shows the nucleotide sequence (SEQ ID NO: 171) and the aminoacid sequence (SEQ ID NO: 38) of the mature heavy chain variable (VH)region of the anti-TIM3 monoclonal antibody 9F6. The CDR1 (SEQ ID NO:45), CDR2 (SEQ ID NO: 50) and CDR3 (SEQ ID NO: 57) are delineated, andthe V, D and J germline derivations are indicated.

FIG. 5B shows the nucleotide sequence (SEQ ID NO: 195) and the aminoacid sequence (SEQ ID NO: 62) of the mature light chain variable (VL)region of VK1 of the anti-TIM3 monoclonal antibody 9F6. The CDR1 (SEQ IDNO: 65), CDR2 (SEQ ID NO: 67) and CDR3 (SEQ ID NO: 70) are delineated,and the V and J germline derivations are indicated.

FIG. 5C shows the nucleotide sequence (SEQ ID NO: 196) and the aminoacid sequence (SEQ ID NO: 63) of the mature light chain variable (VL)region of VK2 of the anti-TIM3 monoclonal antibody 9F6. The CDR1 (SEQ IDNO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 71) are delineated,and the V and J germline derivations are indicated.

FIG. 5D shows the nucleotide sequence (SEQ ID NO: 194) and the aminoacid sequence (SEQ ID NO: 61) of the mature light chain variable (VL)region of VK3 of the anti-TIM3 monoclonal antibody 9F6. The CDR1 (SEQ IDNO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are delineated,and the V and J germline derivations are indicated.

FIG. 5E shows the nucleotide sequence (SEQ ID NO: 171) and the aminoacid sequence (SEQ ID NO: 38) of the heavy chain VH region of theanti-TIM3 monoclonal antibody 9F6 with a signal sequence (SEQ ID NOs:275 and 270, respectively), and the nucleotide sequences (SEQ ID NO:195, 196, and 194, respectively) and the amino acid sequences (SEQ IDNO: 62, 63, and 61, respectively) of the light chain VL region of VK1,VK2 and VK3 of the anti-TIM3 monoclonal antibody 9F6 with a signalsequence (SEQ ID NOs: 276 and 271, respectively).

FIG. 6A shows the nucleotide sequence (SEQ ID NO: 172) and the aminoacid sequence (SEQ ID NO: 39) of the mature heavy chain variable (VH)region of the anti-TIM3 monoclonal antibody 3G4. The CDR1 (SEQ ID NO:45), CDR2 (SEQ ID NO: 51) and CDR3 (SEQ ID NO: 58) are delineated, andthe V, D and J germline derivations are indicated.

FIG. 6B shows the nucleotide sequence (SEQ ID NO: 193) and the aminoacid sequence (SEQ ID NO: 60) of the mature light chain variable (VL)region of the anti-TIM3 monoclonal antibody 3G4. The CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 68) are delineated, andthe V and J germline derivations are indicated.

FIG. 6C shows the nucleotide sequence (SEQ ID NO: 172) and the aminoacid sequence (SEQ ID NO: 39) of the heavy chain VH region of theanti-TIM3 monoclonal antibody 3G4 with a signal sequence (SEQ ID NOs:275 and 270, respectively), and the nucleotide sequence (SEQ ID NO: 193)and the amino acid sequence (SEQ ID NO: 60) of the light chain VL regionof the anti-TIM3 monoclonal antibody 3G4 with a signal sequence (SEQ IDNOs: 273 and 268, respectively).

FIG. 7A shows the nucleotide sequence (SEQ ID NO: 173) and the aminoacid sequence (SEQ ID NO: 40) of the mature heavy chain variable (VH)region of the anti-TIM3 monoclonal antibody 17C8. The CDR1 (SEQ ID NO:45), CDR2 (SEQ ID NO: 52) and CDR3 (SEQ ID NO: 59) are delineated, andthe V, D and J germline derivations are indicated.

FIG. 7B shows the nucleotide sequence (SEQ ID NO: 194) and the aminoacid sequence (SEQ ID NO: 61) of the mature light chain variable (VL)region of the anti-TIM3 monoclonal antibody 17C8. The CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are delineated, andthe V and J germline derivations are indicated.

FIG. 7C shows the nucleotide sequence (SEQ ID NO: 173) and the aminoacid sequence (SEQ ID NO: 40) of the heavy chain VH region of theanti-TIM3 monoclonal antibody 17C8 with a signal sequence (SEQ ID NOs:275 and 270, respectively), and the nucleotide sequence (SEQ ID NO: 194)and the amino acid sequence (SEQ ID NO: 61) of the light chain VL regionof the anti-TIM3 monoclonal antibody 17C8 with a signal sequence (SEQ IDNOs: 273 and 268, respectively).

FIG. 8A shows a sequence alignment of the heavy chain variable (VH)region of monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, and17C8. The complementarity determining regions (CDRs) are boxed.

FIG. 8B lists the SEQ ID NOs for the VH regions, each of the CDRs, andmutants thereof, of the antibodies.

FIG. 9A shows a sequence alignment of the light chain variable (VL)region of monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6_VK1, 9F6_VK2,9F6_VK3, 3G4, and 17C8. The complementarity determining regions (CDRs)are boxed.

FIG. 9B lists the SEQ ID NOs for the VL regions and each of the CDRs ofthe antibodies.

FIG. 10 shows a sequence alignment of the mature full length heavy chain(HC) of monoclonal antibody TIM3.5 (13A3) and exemplary variantsthereof: TIM3.13 (D101E), TIM3.14 (P102V), TIM3.15 (P102Y), TIM3.16(P102L), TIM3.17 (N60Q/P102Y), TIM3.18 (N60Q/D101E), TIM3.10 (N60Q),TIM3.11 (N60S), and TIM3.12 (N60A). The VH region of each of the heavychains is underlined.

FIG. 11 shows a sequence alignment of the mature full length HC ofmonoclonal antibody 9F6 and an exemplary variant TIM3.7 (A108T) thereof.The VH region of each heavy chain is underlined.

FIG. 12 shows a sequence alignment of the mature full length HC ofmonoclonal 8B9 and an exemplary variant TIM3.8 (S61P) thereof. The VHregion of each heavy chain is underlined.

FIG. 13 lists the SEQ ID NOs of the full length heavy and light chains,variable regions and CDRs of hybridoma derived antibodies (13A3, 8B9,8C4, 17C3, 9F6, 3G4 and 17C8) and recombinant (TIM3.2-TIM3.18)anti-human TIM3 antibodies. The isotype of the heavy and light chains isalso indicated. “H.n.” refers to hybridoma name. Heavy and light chainsthat are referred to in FIG. 13 can be derived from its elements, e.g.,variable and constant regions that are disclosed herein. Where a SEQ IDNO does not appear in a given column on the second or third page of thetable, it is provided in that column in the page preceding it or thepage preceding that one.

FIGS. 14A-14B show the binding curves and EC₅₀s of anti-TIM3 antibodiesto human TIM-3 transfected CHO cells (FIG. 14A) and activated human Tcells (FIG. 14B).

FIGS. 15A-15B show the binding curves and EC₅₀s of anti-TIM3 antibodiesto a cyno TIM3-transfected CHO cell line (FIG. 15A) and activated cyno Tcells (FIG. 15B).

FIG. 16 shows anti-TIM3 activity (at various antibody concentrations) inpromoting IFN-γ production from tumor infiltrating leukocytes (TILs) inrenal cell carcinoma (RCC). The 8 bars for each antibody representdifferent concentrations of antibody, as indicated.

FIGS. 17A-17B show anti-TIM3 activity (at various antibodyconcentrations) in promoting IFN-γ production from lung cancer TILs(FIG. 17A, IFN-γ ELISA; FIG. 17B, intracellular IFN-γ staining). In FIG.17A, the individual bar for each antibody represents differentconcentrations of antibody, as indicated. In FIG. 17B, the upper panelshows CD4⁺ cells and the lower panel shows CD8⁺ cells. The level of TIM3was measured with 8B9 (x-axis).

FIG. 18 shows anti-TIM-3 antibodies (i.e., antibodies 13A3 and 3G4) inpromoting IFN-γ secretion from TILs isolated from various tissues in thepresence of CHO-OKT3 cells.

FIG. 19 shows anti-TIM-3 cross-blocking of TIM-3 antibodies on activatedhuman T cells.

FIG. 20 shows the amino acid residues that are necessary for binding ofanti-TIM3 monoclonal antibodies 13A3, 3G4, 17C3 and 8B9 to human TIM3.The signal sequence and the transmembrane domains are underlined.

FIGS. 21A-21B show that certain anti-TIM3 antibodies block theinteraction between human TIM3 and PS-liposome. FIG. 21A shows aschematic diagram of the phosphatidylserine (PS)-hTIM3 “in-tandem”blocking assay. FIG. 21B shows blocking of binding of hTIM3-Fc toPS-liposome by certain anti-TIM3 antibodies, as measured via thePS-hTIM3 “in-tandem” blocking assay shown in FIG. 21A.

FIG. 22 shows a summary of the functional activity of various anti-TIM3antibodies (e.g., TIM3.5, TIM3.4, TIM3.2, TIM3.9, 9F6, TIM3.8, andTIM3.6). Data for the binding assay, T-cell assay, TIL assay, andPS-TIM3 blocking assay are provided.

FIG. 23 provides a listing of all SEQ ID Numbers with a description ofthe sequences represented by the SEQ ID Numbers.

FIGS. 24A-24B show the anti-tumor activity of the combinedadministration of anti-PD 1 and anti-TIM3 antibodies in the CT26colorectal tumor mouse model. FIG. 24A shows the tumor volume at varioustime points post tumor implantation in mice (n=10/group) treated withthe (i) control IgG (upper left panel), (ii) RMT3-23 anti-TIM3 antibodyalone (upper right panel), (iii) RMP1-14 anti-PD 1 antibody alone(bottom left panel), and (iv) combination of the RMT3-23 anti-TIM3 andRMP1-14 anti-PD 1 antibodies (bottom right panel). FIG. 24B shows theaverage tumor volume as a function of time (days post tumorimplantation) in mice treated with (i) RMT3-23 anti-TIM3 antibody alone,(ii) AbM anti-TIM3 antibody alone, (iii) RMP1-14 anti-PD 1 antibodyalone, (iv) combination of RMT3-23 anti-TIM3 and RMPP1-14 anti-PD 1antibodies, (v) combination of Ab M anti-TIM3 and RMP1-14 anti-PD 1antibodies, and (vi) isotype control antibody.

FIG. 25 shows the list of common peptides of hTIM-3 that were used tomap the epitopes of the anti-TIM3 antibodies (13A3 and 3G4) usinghydrogen/deuterium exchange mass spectrometry (HDX-MS). Each barindicates a peptic peptide. The circled residues (i.e., N99, T145, andN172) indicate the glycosylation sites.

FIG. 26 shows the human TIM-3 binding regions of the anti-TIM3antibodies (13A3 and 3G4) identified using HDX-MX. The upper panel showsthe binding region of the 13A3 anti-TIM3 antibody. The bottom panelshows the binding region of the 3G4 anti-TIM3 antibody.

FIGS. 27A-27B show the results of a Scatchard analysis of TIM3.18.IgG1.3to CHO cells ectopically expressing human or cyno TIM3. FIG. 27A shows a¹²⁵I-TIM3 Ab standard curve. FIG. 27B shows the amount of TIM3.18.IgG1.3antibody bound to CHO cells expressing human (left panel) and cyno(right panel) TIM3.

FIG. 28 shows the results of a Scatchard analysis of TIM3.18.IgG1.3 toactivated Th1 cells from two donors (left and right panels).

FIGS. 29A and 29B show TIM3.18.IgG1.3 and TIM3.18.IgG1.3 Fab enhancedproliferation of Th1 T cells in the polarized Th1/irradiated CHO-OKT3co-culture assay. FIG. 29A shows Th1 cell proliferation observed withvarious concentrations of TIM3.18.IgG1.3, 13A3 (“13A3-g4”) or with noantibody or isotype control antibodies (hIgG1.1 and hIgG4). FIG. 29Bshows Th1 cell proliferation observed with various concentrations ofTIM3.18.IgG1.3 Fab or with no antibody or isotype control antibodyIgG1.3.

FIG. 30 shows that anti-TIM3 antibody TIM3.18.IgG1.3 enhancedproliferation of Th1 T cells in the polarized Th1/irradiatedCHO-OKT3-PD-L1 co-culture assay in combination with nivolumab.

FIG. 31 shows that anti-TIM3 antibody TIM3.18.IgG1.3 enhancedinterferon-γ secretion of renal cell carcinoma tumor infiltratinglymphocytes (TILs) stimulated with irradiated CHO-OKT3 cells.

FIG. 32 shows that anti-TIM3 antibody TIM3.18.IgG1.3 enhancedinterferon-γ secretion of breast cancer TILs stimulated with irradiatedCHO-OKT3 cells.

FIG. 33 shows CD163, CD206 and TIM3 expression on the M0 macrophagesthat were used in an AlloMLR (mixed lymphocyte reaction) assay, theresults of which are shown in FIG. 34.

FIG. 34 shows the proliferation of cells in an AlloMLR assay conductedin the presence of the anti-TIM3 antibody TIM3.18.IgG1.3, an isotypecontrol or in the absence of antibody.

FIG. 35 is a ribbon diagram of the structure of a TIM3:TIM3.18 Fabcomplex, as determined by crystallography. The Fab fragment is shown inlight gray and TIM3 is shown in dark gray.

FIG. 36 shows the structure of TIM3:TIM3.18 Fab complex, as determinedby crystallography. The Fab fragment is shown as a ribbon diagram. TIM3is shown in white surface representation, with the Fab contact residuesdepicted in dark gray.

FIG. 37 is a diagram of the assay that was used to measure potentialinternalization by anti-TIM3 antibodies.

FIG. 38 shows that anti-TIM3 antibodies 13A3 (bottom left panel) andcertain variants (D101E—top left panel; N60Q—top right panel) thereof donot trigger receptor (i.e., TIM3) mediated internalization.

FIGS. 39A and 39 B show a ribbon diagram depicting the epitopes ofanti-TIM3 antibodies 13A3 (FIG. 39A) and 3G4 (FIG. 39B). The amino acidsequences of the epitopes for each of the antibodies are provided belowthe ribbon diagram. The different patterns identify the specific regionsof the anti-TIM3 antibodies that correspond to the specific epitopes.

DETAILED DESCRIPTION OF DISCLOSURE

In order that the present description can be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleotide sequence,” is understood torepresent one or more nucleotide sequences. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, nucleotidesequences are written left to right in 5′ to 3′ orientation. Amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects ofthe disclosure, which can be had by reference to the specification as awhole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

The term “about” is used herein to mean approximately, roughly, around,or in the regions of. When the term “about” is used in conjunction witha numerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” can modify a numerical value above and below the stated value bya variance of, e.g., 10 percent, up or down (higher or lower).

The term “T-cell immunoglobulin and mucin-domain containing-3” or “TIM3”as used herein refers to a receptor that is a member of the T cellimmunoglobulin and mucin domain (TIM) family of proteins. Primary ligandfor TIM3 include phosphatidylserine (TIM3-L). TIM3 is also referred toas hepatitis A virus cellular receptor 2 (HAVCR2), T-cell immunoglobulinmucin receptor 3, TIM-3, TIMD3, TIMD-3, Kidney Injury Molecule-3, KIM-3,and CD366. The term “TIM3” includes any variants or isoforms of TIM3which are naturally expressed by cells. Accordingly, antibodiesdescribed herein can cross-react with TIM3 from species other than human(e.g., cynomolgus TIM3). Alternatively, the antibodies can be specificfor human TIM3 and do not exhibit any cross-reactivity with otherspecies. TIM3 or any variants and isoforms thereof, can either beisolated from cells or tissues which naturally express them or berecombinantly produced using well-known techniques in the art and/orthose described herein.

Two isoforms of human TIM3 have been identified. Isoform 1 (AccessionNo. NP_116171; SEQ ID NO: 286) consists of 301 amino acids andrepresents the canonical sequence. Isoform 2 (Accession No. AAH20843;SEQ ID NO: 287) consists of 142 amino acids, and is soluble. It lacksamino acid residues 143-301, which encode the transmembrane domain, thecytoplasmic domain, and part of the extracellular domain of TIM3. Theamino acid residues 132-142 also differ from the canonical sequencedescribed above.

Below are the amino acid sequences of the two known human TIM3 isoforms.

(A) Human TIM3 isoform 1 (Accession No. NP_116171; SEQ ID NO: 286;encoded by the nucleotide sequence having Accession No. NM_032782.4; SEQID NO: 288; FIG. 20):

MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAM P(B) Human TIM3 isoform 2 (Accession No. AAH20843; SEQ ID NO: 287;encoded by the nucleotide sequence having Accession No. BC020843.1; SEQID NO: 289):

MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPGEWTFACHLYE

The signal sequence of isoforms 1 and 2 corresponds to amino acids 1-21(underlined). Thus, the mature isoforms 1 and 2 consist of amino acids22 to 301 or 142, respectively. The extracellular domain of mature humanTIM3 consists of amino acids 22-202 of SEQ ID NO: 286 and has the aminoacid sequence:

(SEQ ID NO: 290) SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIG.

Cynomolgus TIM3 protein consists of the following amino acid sequence(including a signal sequence):

(SEQ ID NO: 360) MFSHLPFDCVLLLLLLLLTRSSEVEYIAEVGQNAYLPCSYTPAPPGNLVPVCWGKGACPVFDCSNVVLRTENRDVNDRTSGRYWLKGDFHKGDVSLTIENVTLADSGVYCCRIQIPGIMNDEKHNLKLVVIKPAKVTPAPTLQRDLTSAFPRMLTTGEHGPAETQTPGSLPDVNLTQIFTLTNELRDSGATIRTAIYIAAGISAGLALALIFGALIFKWYSHSKEKTQNLSLISLANIPPSGLANAVAEGIRSEENIYTIEEDVYEVEEPNEYYCYVSSGQQPSQPLGCRFAMP

The term “antibody” refers, in one embodiment, to a protein comprisingat least two heavy (H) chains and two light (L) chains inter-connectedby disulfide bonds. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion (abbreviated herein as CH). In certain antibodies, e.g.,naturally occurring IgG antibodies, the heavy chain constant region iscomprised of a hinge and three domains, CH1, CH2 and CH3. In certainantibodies, e.g., naturally occurring IgG antibodies, each light chainis comprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain (abbreviated herein as CL). The VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies can mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system. A heavy chain may have theC-terminal lysine or not. Unless specified otherwise herein, the aminoacids in the variable regions are numbered using the Kabat numberingsystem and those in the constant regions are numbered using the EUsystem.

An “IgG antibody”, e.g., a human IgG1, IgG2, IgG3 and IgG4 antibody, asused herein has, in certain embodiments, the structure of a naturallyoccurring IgG antibody, i.e., it has the same number of heavy and lightchains and disulfide bonds as a naturally occurring IgG antibody of thesame subclass. For example, an anti-TIM3 IgG1, IgG2, IgG3 or IgG4antibody consists of two heavy chains (HCs) and two light chains (LCs),wherein the two heavy chains and light chains are linked by the samenumber and location of disulfide bridges that occur in naturallyoccurring IgG1, IgG2, IgG3 and IgG4 antibodies, respectively (unless theantibody has been mutated to modify the disulfide bridges).

Antibodies typically bind specifically to their cognate antigen withhigh affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁵ to10⁻¹¹ M or less. Any K_(D) greater than about 10⁻⁴ M is generallyconsidered to indicate nonspecific binding. As used herein, an antibodythat “binds specifically” to an antigen refers to an antibody that bindsto the antigen and substantially identical antigens with high affinity,which means having a K_(D) of 10⁻⁷ M or less, 10⁻⁸ M or less, 5×10⁻⁹ Mor less, or between 10⁻⁸ M and 10⁻¹⁰ M or less, but does not bind withhigh affinity to unrelated antigens. An antigen is “substantiallyidentical” to a given antigen if it exhibits a high degree of sequenceidentity to the given antigen, for example, if it exhibits at least 80%,at least 90%, at least 95%, at least 97%, or at least 99% sequenceidentity to the sequence of the given antigen. By way of example, anantibody that binds specifically to human TIM3 can, in certainembodiments, also have cross-reactivity with TIM3 antigens from certainprimate species (e.g., cynomolgus TIM3), but cannot cross-react withTIM3 antigens from other species or with an antigen other than TIM3.

An immunoglobulin can be from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. The IgGisotype is divided in subclasses in certain species: IgG1, IgG2, IgG3and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice. In certainembodiments, the anti-TIM3 antibodies described herein are of the IgG1subtype. Immunoglobulins, e.g., IgG1, exist in several allotypes, whichdiffer from each other in at most a few amino acids. “Antibody”includes, by way of example, both naturally occurring and non-naturallyoccurring antibodies; monoclonal and polyclonal antibodies; chimeric andhumanized antibodies; human and nonhuman antibodies and wholly syntheticantibodies.

The term “antigen-binding portion” of an antibody, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen (e.g., human TIM3). It has been shownthat the antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibody,e.g., an anti-TIM3 antibody described herein, include (i) a Fab fragment(fragment from papain cleavage) or a similar monovalent fragmentconsisting of the V_(L), V_(H), LC and CH1 domains; (ii) a F(ab′)2fragment (fragment from pepsin cleavage) or a similar bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and CH1 domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546), which consists of a V_(H) domain; (vi) an isolatedcomplementarity determining region (CDR) and (vii) a combination of twoor more isolated CDRs which can optionally be joined by a syntheticlinker. Furthermore, although the two domains of the Fv fragment, V_(L)and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies. Antigen-binding portions can be produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody,” as used herein, refers to an antibodyfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprised in the population are substantiallysimilar and bind the same epitope(s) (e.g., the antibodies display asingle binding specificity and affinity), except for possible variantsthat may arise during production of the monoclonal antibody, suchvariants generally being present in minor amounts. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. The term “human monoclonal antibody” refers to an antibody froma population of substantially homogeneous antibodies that display(s) asingle binding specificity and which has variable and optional constantregions derived from human germline immnunoglobulin sequences. In oneembodiment, human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic non-human animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for humanimmnunoglobulin genes or a hybridoma prepared therefrom, (b) antibodiesisolated from a host cell transformed to express the antibody, e.g.,from a transfectoma, (c) antibodies isolated from a recombinant,combinatorial human antibody library, and (d) antibodies prepared,expressed, created or isolated by any other means that involve splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies comprise variable and constant regions thatutilize particular human germline immnunoglobulin sequences are encodedby the germline genes, but include subsequent rearrangements andmutations which occur, for example, during antibody maturation. As knownin the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117-1125),the variable region contains the antigen binding domain, which isencoded by various genes that rearrange to form an antibody specific fora foreign antigen. In addition to rearrangement, the variable region canbe further modified by multiple single amino acid changes (referred toas somatic mutation or hypermutation) to increase the affinity of theantibody to the foreign antigen. The constant region will change infurther response to an antigen (i.e., isotype switch). Therefore, therearranged and somatically mutated nucleic acid molecules that encodethe light chain and heavy chain immunoglobulin polypeptides in responseto an antigen cannot have sequence identity with the original nucleicacid molecules, but instead will be substantially identical or similar(i.e., have at least 80% identity).

A “human” antibody (HuMAb) refers to an antibody having variable regionsin which both the framework and CDR regions are derived from humangermline immunoglobulin sequences. Furthermore, if the antibody containsa constant region, the constant region also is derived from humangermline immunoglobulin sequences. The anti-TIM3 antibodies describedherein can include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. The terms “human” antibodies and “fully human”antibodies are used synonymously.

A “humanized” antibody refers to an antibody in which some, most or allof the amino acids outside the CDR domains of a non-human antibody arereplaced with corresponding amino acids derived from humanimmunoglobulins. In one embodiment of a humanized form of an antibody,some, most or all of the amino acids outside the CDR domains have beenreplaced with amino acids from human immunoglobulins, whereas some, mostor all amino acids within one or more CDR regions are unchanged. Smalladditions, deletions, insertions, substitutions or modifications ofamino acids are permissible as long as they do not abrogate the abilityof the antibody to bind to a particular antigen. A “humanized” antibodyretains an antigenic specificity similar to that of the originalantibody.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody.

As used herein, “isotype” refers to the antibody class (e.g., IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that isencoded by the heavy chain constant region genes.

“Allotype” refers to naturally occurring variants within a specificisotype group, which variants differ in a few amino acids (see, e.g.,Jefferis et al. (2009) mAbs 1:1). Anti-TIM3 antibodies described hereincan be of any allotype. As used herein, antibodies referred to as“IgG1f,” “IgG1.1f,” or “IgG1.3f” isotype are IgG1, effectorless IgG1.1,and effectorless IgG1.3 antibodies, respectively, of the allotype “f,”i.e., having 214R, 356E and 358M according to the EU index as in Kabat,as shown, e.g., in SEQ ID NO: 3.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other proteins and cellularmaterial.

As used herein, an antibody that “inhibits binding of TIM3-L to TIM3” isintended to refer to an antibody that inhibits the binding of TIM3 toits ligand, e.g., phosphatidylserine, e.g., in binding assays using CHOcells transfected with human TIM3 or TIM3 expressing activated T cells,with an EC₅₀ of about 1 μg/mL or less, such as about 0.9 μg/mL or less,about 0.85 μg/mL or less, about 0.8 μg/mL or less, about 0.75 μg/mL orless, about 0.7 μg/mL or less, about 0.65 μg/mL or less, about 0.6 μg/mLor less, about 0.55 μg/mL or less, about 0.5 μg/mL or less, about 0.45μg/mL or less, about 0.4 μg/mL or less, about 0.35 μg/mL or less, about0.3 μg/mL or less, about 0.25 μg/mL or less, about 0.2 μg/mL or less,about 0.15 μg/mL or less, about 0.1 μg/mL or less, or about 0.05 μg/mLor less, in art-recognized methods, e.g., the FACS-based binding assaysdescribed herein.

An “effector function” refers to the interaction of an antibody Fcregion with an Fc receptor or ligand, or a biochemical event thatresults therefrom. Exemplary “effector functions” include C1q binding,complement dependent cytotoxicity (CDC), Fc receptor binding,FcγR-mediated effector functions such as ADCC and antibody dependentcell-mediated phagocytosis (ADCP), and downregulation of a cell surfacereceptor (e.g., the B cell receptor; BCR). Such effector functionsgenerally require the Fc region to be combined with a binding domain(e.g., an antibody variable domain).

An “Fc receptor” or “FcR” is a receptor that binds to the Fc region ofan immunoglobulin. FcRs that bind to an IgG antibody comprise receptorsof the FcγR family, including allelic variants and alternatively splicedforms of these receptors. The FcγR family consists of three activating(FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA inhumans) and one inhibitory (FcγRIIB) receptor. Various properties ofhuman FcγRs are known in the art. The majority of innate effector celltypes coexpress one or more activating FcγR and the inhibitory FcγRIIB,whereas natural killer (NK) cells selectively express one activating Fcreceptor (FcγRIII in mice and FcγRIIIA in humans) but not the inhibitoryFcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptorsand is considered equivalent to murine IgG2a with respect to the typesof activating Fc receptors that it binds to.

An “Fc region” (fragment crystallizable region) or “Fc domain” or “Fc”refers to the C-terminal region of the heavy chain of an antibody thatmediates the binding of the immunoglobulin to host tissues or factors,including binding to Fc receptors located on various cells of the immunesystem (e.g., effector cells) or to the first component (C1q) of theclassical complement system. Thus, an Fc region comprises the constantregion of an antibody excluding the first constant region immunoglobulindomain (e.g., CH1 or CL). In IgG, IgA and IgD antibody isotypes, the Fcregion comprises two identical protein fragments, derived from thesecond (CH2) and third (CH3) constant domains of the antibody's twoheavy chains; IgM and IgE Fc regions comprise three heavy chain constantdomains (CH domains 2-4) in each polypeptide chain. For IgG, the Fcregion comprises immunoglobulin domains CH2 and CH3 and the hingebetween CH1 and CH2 domains. Although the definition of the boundariesof the Fc region of an immunoglobulin heavy chain might vary, as definedherein, the human IgG heavy chain Fc region is defined to stretch froman amino acid residue D221 for IgG1, V222 for IgG2, L221 for IgG3 andP224 for IgG4 to the carboxy-terminus of the heavy chain, wherein thenumbering is according to the EU index as in Kabat. The CH2 domain of ahuman IgG Fc region extends from amino acid 237 to amino acid 340, andthe CH3 domain is positioned on C-terminal side of a CH2 domain in an Fcregion, i.e., it extends from amino acid 341 to amino acid 447 or 446(if the C-terminal lysine residue is absent) or 445 (if the C-terminalglycine and lysine residues are absent) of an IgG. As used herein, theFc region can be a native sequence Fc, including any allotypic variant,or a variant Fc (e.g., a non-naturally occurring Fc). Fc can also referto this region in isolation or in the context of an Fc-comprisingprotein polypeptide such as a “binding protein comprising an Fc region,”also referred to as an “Fc fusion protein” (e.g., an antibody orimmunoadhesion).

A “native sequence Fc region” or “native sequence Fc” comprises an aminoacid sequence that is identical to the amino acid sequence of an Fcregion found in nature. Native sequence human Fc regions include anative sequence human IgG1 Fc region; native sequence human IgG2 Fcregion; native sequence human IgG3 Fc region; and native sequence humanIgG4 Fc region as well as naturally occurring variants thereof. Nativesequence Fc include the various allotypes of Fcs (see, e.g., Jefferis etal. (2009) mAbs 1: 1).

The term “epitope” or “antigenic determinant” refers to a site on anantigen (e.g., TIM3) to which an immunoglobulin or antibody specificallybinds, e.g., as defined by the specific method used to identify it.Epitopes can be formed both from contiguous amino acids (usually alinear epitope) or noncontiguous amino acids juxtaposed by tertiaryfolding of a protein (usually a conformational epitope). Epitopes formedfrom contiguous amino acids are typically, but not always, retained onexposure to denaturing solvents, whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15 amino acids in a unique spatial conformation. Methods fordetermining what epitopes are bound by a given antibody (i.e., epitopemapping) are well known in the art and include, for example,immunoblotting and immunoprecipitation assays, wherein overlapping orcontiguous peptides from (e.g., from TIM3) are tested for reactivitywith a given antibody (e.g., anti-TIM3 antibody). Methods of determiningspatial conformation of epitopes include techniques in the art and thosedescribed herein, for example, x-ray crystallography, antigen mutationalanalysis, 2-dimensional nuclear magnetic resonance and HDX-MS (see,e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, G. E. Morris, Ed. (1996)).

The term “epitope mapping” refers to the process of identification ofthe molecular determinants for antibody-antigen recognition.

The term “binds to the same epitope” with reference to two or moreantibodies means that the antibodies bind to the same segment of aminoacid residues, as determined by a given method. Techniques fordetermining whether antibodies bind to the “same epitope on TIM3” withthe antibodies described herein include, for example, epitope mappingmethods, such as, x-ray analyses of crystals of antigen:antibodycomplexes which provides atomic resolution of the epitope andhydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methodsmonitor the binding of the antibody to antigen fragments or mutatedvariations of the antigen where loss of binding due to a modification ofan amino acid residue within the antigen sequence is often considered anindication of an epitope component. In addition, computationalcombinatorial methods for epitope mapping can also be used. Thesemethods rely on the ability of the antibody of interest to affinityisolate specific short peptides from combinatorial phage display peptidelibraries. Antibodies having the same VH and VL or the same CDR1, 2 and3 sequences are expected to bind to the same epitope.

Antibodies that “compete with another antibody for binding to a target”refer to antibodies that inhibit (partially or completely) the bindingof the other antibody to the target. Whether two antibodies compete witheach other for binding to a target, i.e., whether and to what extent oneantibody inhibits the binding of the other antibody to a target, can bedetermined using known competition experiments, e.g., BIACORE® surfaceplasmon resonance (SPR) analysis. In certain embodiments, an antibodycompetes with, and inhibits binding of another antibody to a target byat least 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition orcompetition can be different depending on which antibody is the“blocking antibody” (i.e., the cold antibody that is incubated firstwith the target). Competition assays can be conducted as described, forexample, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006;doi: 10.1101/pdb.prot4277 or in Chapter 11 of “Using Antibodies” by EdHarlow and David Lane, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., USA 1999. Two antibodies “cross-compete” if antibodiesblock each other both ways by at least 50%, i.e., regardless of whetherone or the other antibody is contacted first with the antigen in thecompetition experiment.

Competitive binding assays for determining whether two antibodiescompete or cross-compete for binding include: competition for binding toT cells expressing TIM3, e.g., by flow cytometry, such as described inthe Examples. Other methods include: SPR (e.g., BIACORE®), solid phasedirect or indirect radioimmunoassay (RIA), solid phase direct orindirect enzyme immunoassay (EIA), sandwich competition assay (seeStahli et al., Methods in Enzymology 9:242 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986));solid phase direct labeled assay, solid phase direct labeled sandwichassay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Press (1988)); solid phase direct label RIA using 1-125 label(see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase directbiotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and directlabeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen. Typically, the antibody (i)binds with an equilibrium dissociation constant (K_(D)) of approximatelyless than 10⁻⁷ M, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or10⁻¹⁰ M or even lower when determined by, e.g., surface plasmonresonance (SPR) technology in a BIACORE® 2000 instrument using thepredetermined antigen, e.g., recombinant human TIM3, as the analyte andthe antibody as the ligand, or Scatchard analysis of binding of theantibody to antigen positive cells, and (ii) binds to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.Accordingly, an antibody that “specifically binds to human TIM3” refersto an antibody that binds to soluble or cell bound human TIM3 with aK_(D) of 10⁻⁷ M or less, such as approximately less than 10⁻⁸ M, 10⁻⁹ Mor 10⁻¹⁰ M or even lower. An antibody that “cross-reacts with cynomolgusTIM3” refers to an antibody that binds to cynomolgus TIM3 with a K_(D)of 10⁻⁷ M or less, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or10⁻¹⁰ M or even lower. In certain embodiments, such antibodies that donot cross-react with TIM3 from a non-human species exhibit essentiallyundetectable binding against these proteins in standard binding assays.

The term “k_(assoc)” or “k_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “k_(dis)” or “k_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of k_(d) tok_(a) (i.e., k_(d)/k_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. Available methods for determining the K_(D) ofan antibody include surface plasmon resonance, a biosensor system suchas a BIACORE® system or flow cytometry and Scatchard analysis.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, 10⁻⁹M or less, or 10⁻¹ Mor less for a target antigen. However, “high affinity” binding can varyfor other antibody isotypes. For example, “high affinity” binding for anIgM isotype refers to an antibody having a K_(D) of 10⁻¹⁰ M or less, or10⁻⁸ M or less.

The term “EC₅₀” in the context of an in vitro or in vivo assay using anantibody or antigen binding fragment thereof, refers to theconcentration of an antibody or an antigen-binding portion thereof thatinduces a response that is 50% of the maximal response, i.e., halfwaybetween the maximal response and the baseline.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

A “polypeptide” refers to a chain comprising at least two consecutivelylinked amino acid residues, with no upper limit on the length of thechain. One or more amino acid residues in the protein can contain amodification such as, but not limited to, glycosylation, phosphorylationor disulfide bond formation. A “protein” can comprise one or morepolypeptides.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule can besingle-stranded or double-stranded, and can be cDNA.

“Conservative amino acid substitutions” refer to substitutions of anamino acid residue with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). In certainembodiments, a predicted nonessential amino acid residue in an anti-TIM3antibody is replaced with another amino acid residue from the same sidechain family. Methods of identifying nucleotide and amino acidconservative substitutions which do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999);and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, at least about 90%to 95%, or at least about 98% to 99.5% of the nucleotides.Alternatively, substantial homology exists when the segments willhybridize under selective hybridization conditions, to the complement ofthe strand.

For polypeptides, the term “substantial homology” indicates that twopolypeptides, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate amino acid insertions ordeletions, in at least about 80% of the amino acids, at least about 90%to 95%, or at least about 98% to 99.5% of the amino acids.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available atworldwideweb.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences described herein can further beused as a “query sequence” to perform a search against public databasesto, for example, identify related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules described herein. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See worldwideweb.ncbi.nlm.nih.gov.

The nucleic acids can be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids (e.g., the other parts of the chromosome) or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York (1987).

Nucleic acids, e.g., cDNA, can be mutated, in accordance with standardtechniques to provide gene sequences. For coding sequences, thesemutations, can affect amino acid sequence as desired. In particular, DNAsequences substantially homologous to or derived from native V, D, J,constant, switches and other such sequences described herein arecontemplated (where “derived” indicates that a sequence is identical ormodified from another sequence).

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”) In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, also included are other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell that comprises a nucleic acidthat is not naturally present in the cell, and can be a cell into whicha recombinant expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications can occur in succeeding generations due to eithermutation or environmental influences, such progeny cannot, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

An “immune response” is as understood in the art, and generally refersto a biological response within a vertebrate against foreign agents orabnormal, e.g., cancerous cells, which response protects the organismagainst these agents and diseases caused by them. An immune response ismediated by the action of one or more cells of the immune system (forexample, a T lymphocyte, B lymphocyte, natural killer (NK) cell,macrophage, eosinophil, mast cell, dendritic cell or neutrophil) andsoluble macromolecules produced by any of these cells or the liver(including antibodies, cytokines, and complement) that results inselective targeting, binding to, damage to, destruction of, and/orelimination from the vertebrate's body of invading pathogens, cells ortissues infected with pathogens, cancerous or other abnormal cells, or,in cases of autoimmunity or pathological inflammation, normal humancells or tissues. An immune reaction includes, e.g., activation orinhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4+cell, a CD8+ T cell, or a Treg cell, or activation or inhibition of anyother cell of the immune system, e.g., NK cell.

An “immunomodulator” or “immunoregulator” refers to an agent, e.g., anagent targeting a component of a signaling pathway that can be involvedin modulating, regulating, or modifying an immune response.“Modulating,” “regulating,” or “modifying” an immune response refers toany alteration in a cell of the immune system or in the activity of suchcell (e.g., an effector T cell, such as a Th1 cell). Such modulationincludes stimulation or suppression of the immune system which can bemanifested by an increase or decrease in the number of various celltypes, an increase or decrease in the activity of these cells, or anyother changes which can occur within the immune system. Both inhibitoryand stimulatory immunomodulators have been identified, some of which canhave enhanced function in a tumor microenvironment. In some embodiments,the immunomodulator targets a molecule on the surface of a T cell. An“immunomodulatory target” or “immunoregulatory target” is a molecule,e.g., a cell surface molecule, that is targeted for binding by, andwhose activity is altered by the binding of, a substance, agent, moiety,compound or molecule. Immunomodulatory targets include, for example,receptors on the surface of a cell (“immunomodulatory receptors”) andreceptor ligands (“immunomodulatory ligands”).

“Immunotherapy” refers to the treatment of a subject afflicted with, orat risk of contracting or suffering a recurrence of, a disease by amethod comprising inducing, enhancing, suppressing or otherwisemodifying the immune system or an immune response.

“Immuno stimulating therapy” or “immuno stimulatory therapy” refers to atherapy that results in increasing (inducing or enhancing) an immuneresponse in a subject for, e.g., treating cancer.

“Potentiating an endogenous immune response” means increasing theeffectiveness or potency of an existing immune response in a subject.This increase in effectiveness and potency can be achieved, for example,by overcoming mechanisms that suppress the endogenous host immuneresponse or by stimulating mechanisms that enhance the endogenous hostimmune response.

“T effector” (“T_(eff)”) cells refers to T cells (e.g., CD4+ and CD8+ Tcells) with cytolytic activities as well as T helper (Th) cells, e.g.,Th1 cells, which cells secrete cytokines and activate and direct otherimmune cells, but does not include regulatory T cells (Treg cells).Certain anti-TIM3 antibodies described herein activate T_(eff) cells,e.g., CD4+ and CD8+T_(eff) cells and Th1 cells.

An increased ability to stimulate an immune response or the immunesystem, can result from an enhanced agonist activity of T cellco-stimulatory receptors and/or an enhanced antagonist activity ofinhibitory receptors. An increased ability to stimulate an immuneresponse or the immune system can be reflected by a fold increase of theEC₅₀ or maximal level of activity in an assay that measures an immuneresponse, e.g., an assay that measures changes in cytokine or chemokinerelease, cytolytic activity (determined directly on target cells orindirectly via detecting CD107a or granzymes) and proliferation. Theability to stimulate an immune response or the immune system activitycan be enhanced by at least 10%, 30%, 50%, 75%, 2 fold, 3 fold, 5 foldor more.

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (i.e., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical conjugation and recombinant protein production.

As used herein, “administering” refers to the physical introduction of acomposition comprising a therapeutic agent to a subject, using any ofthe various methods and delivery systems known to those skilled in theart. Different routes of administration for the anti-TIM3 antibodiesdescribed herein include intravenous, intraperitoneal, intramuscular,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intraperitoneal, intramuscular, intraarterial,intrathecal, intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

As used herein, the term “T cell-mediated response” refers to a responsemediated by T cells, including effector T cells (e.g., CD8⁺ cells) andhelper T cells (e.g., CD4⁺ cells). T cell mediated responses include,for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8⁺ T cells.

As used herein, the terms “inhibits” or “blocks” (e.g., referring toinhibition/blocking of binding of TIM3-L to TIM3 on cells) are usedinterchangeably and encompass both partial and completeinhibition/blocking. In some embodiments, the anti-TIM3 antibodyinhibits binding of TIM3-L to TIM3 by at least about 50%, for example,about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, determined, e.g., asfurther described herein. In some embodiments, the anti-TIM3 antibodyinhibits binding of TIM3-L to TIM3 by no more than 50%, for example, byabout 40%, 30%, 20%, 10%, 5% or 1%, determined, e.g., as furtherdescribed herein.

As used herein, the phrase “inhibits growth of a tumor” includes anymeasurable decrease in the growth of a tumor, e.g., the inhibition ofgrowth of a tumor by at least about 10%, for example, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 99%, or 100%.

As used herein, “cancer” refers a broad group of diseases characterizedby the uncontrolled growth of abnormal cells in the body. Unregulatedcell division can result in the formation of malignant tumors or cellsthat invade neighboring tissues and can metastasize to distant parts ofthe body through the lymphatic system or bloodstream.

The terms “treat,” “treating,” and “treatment,” as used herein, refer toany type of intervention or process performed on, or administering anactive agent to, the subject with the objective of reversing,alleviating, ameliorating, inhibiting, or slowing down or preventing theprogression, development, severity or recurrence of a symptom,complication, condition or biochemical indicia associated with a diseaseor enhancing overall survival. Treatment can be of a subject having adisease or a subject who does not have a disease (e.g., forprophylaxis).

A “hematological malignancy” includes a lymphoma, leukemia, myeloma or alymphoid malignancy, as well as a cancer of the spleen and the lymphnodes. Exemplary lymphomas include both B cell lymphomas (a B-cellhematological cancer) and T cell lymphomas. B-cell lymphomas includeboth Hodgkin's lymphomas and most non-Hodgkin's lymphomas. Non-limitingexamples of B cell lymphomas include diffuse large B-cell lymphoma,follicular lymphoma, mucosa-associated lymphatic tissue lymphoma, smallcell lymphocytic lymphoma (overlaps with chronic lymphocytic leukemia),mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B celllymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B celllymphoma, splenic marginal zone lymphoma, intravascular large B-celllymphoma, primary effusion lymphoma, lymphomatoid granulomatosis.Non-limiting examples of T cell lymphomas include extranodal T celllymphoma, cutaneous T cell lymphomas, anaplastic large cell lymphoma,and angioimmunoblastic T cell lymphoma. Hematological malignancies alsoinclude leukemia, such as, but not limited to, secondary leukemia,chronic lymphocytic leukemia, acute myelogenous leukemia, chronicmyelogenous leukemia, and acute lymphoblastic leukemia. Hematologicalmalignancies further include myelomas, such as, but not limited to,multiple myeloma and smoldering multiple myeloma. Other hematologicaland/or B cell- or T-cell-associated cancers are encompassed by the termhematological malignancy.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve a desired effect. A“therapeutically effective amount” or “therapeutically effective dosage”of a drug or therapeutic agent is any amount of the drug that, when usedalone or in combination with another therapeutic agent, promotes diseaseregression evidenced by a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction. Atherapeutically effective amount or dosage of a drug includes a“prophylactically effective amount” or a “prophylactically effectivedosage”, which is any amount of the drug that, when administered aloneor in combination with another therapeutic agent to a subject at risk ofdeveloping a disease or of suffering a recurrence of disease, inhibitsthe development or recurrence of the disease. The ability of atherapeutic agent to promote disease regression or inhibit thedevelopment or recurrence of the disease can be evaluated using avariety of methods known to the skilled practitioner, such as in humansubjects during clinical trials, in animal model systems predictive ofefficacy in humans, or by assaying the activity of the agent in in vitroassays.

By way of example, an anti-cancer agent is a drug that promotes cancerregression in a subject. In some embodiments, a therapeuticallyeffective amount of the drug promotes cancer regression to the point ofeliminating the cancer. “Promoting cancer regression” means thatadministering an effective amount of the drug, alone or in combinationwith an antineoplastic agent, results in a reduction in tumor growth orsize, necrosis of the tumor, a decrease in severity of at least onedisease symptom, an increase in frequency and duration of diseasesymptom-free periods, a prevention of impairment or disability due tothe disease affliction, or otherwise amelioration of disease symptoms inthe patient. In addition, the terms “effective” and “effectiveness” withregard to a treatment includes both pharmacological effectiveness andphysiological safety. Pharmacological effectiveness refers to theability of the drug to promote cancer regression in the patient.Physiological safety refers to the level of toxicity, or other adversephysiological effects at the cellular, organ and/or organism level(adverse effects) resulting from administration of the drug.

By way of example for the treatment of tumors, a therapeuticallyeffective amount or dosage of the drug inhibits cell growth or tumorgrowth by at least about 20%, by at least about 40%, by at least about60%, or by at least about 80% relative to untreated subjects. In someembodiments, a therapeutically effective amount or dosage of the drugcompletely inhibits cell growth or tumor growth, i.e., inhibits cellgrowth or tumor growth by 100%. The ability of a compound to inhibittumor growth can be evaluated using the assays described infra.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit cell growth, suchinhibition can be measured in vitro by assays known to the skilledpractitioner. In other embodiments described herein, tumor regressioncan be observed and continue for a period of at least about 20 days, atleast about 40 days, or at least about 60 days.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions described herein canbe used to treat a subject having cancer. The term “non-human animal”includes all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, reptiles,etc.

The term “weight based” dose or dosing as referred to herein means thata dose that is administered to a patient is calculated based on theweight of the patient. For example, when a patient with 60 kg bodyweight requires 3 mg/kg of an anti-TIM3 antibody, one can calculate anduse the appropriate amount of the anti-TIM3 antibody (i.e., 180 mg) foradministration.

The use of the term “fixed dose” with regard to a method of thedisclosure means that two or more different antibodies in a singlecomposition (e.g., anti-TIM3 antibody and a second antibody, e.g., aPD-1 or PD-L1 antibody) are present in the composition in particular(fixed) ratios with each other. In some embodiments, the fixed dose isbased on the weight (e.g., mg) of the antibodies. In certainembodiments, the fixed dose is based on the concentration (e.g., mg/ml)of the antibodies. In some embodiments, the ratio of the two antibodies(e.g., anti-TIM3 and anti-PD1 or anti-PD-L1) is at least about 1:1,about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about1:100, about 1:120, about 1:140, about 1:160, about 1:180, about 1:200,about 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about40:1, about 30:1, about 20:1, about 15:1, about 10:1, about 9:1, about8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1mg first antibody (e.g., anti-TIM3 antibody) to mg second antibody. Forexample, a 2:1 ratio of an anti-TIM3 antibody and a PD-1 antibody, suchas nivolumab, can mean that a vial or an injection can contain about 480mg of the anti-TIM3 antibody and 240 mg of the anti-PD-1 antibody, orabout 2 mg/ml of the anti-TIM3 antibody and 1 mg/ml of the anti-PD-1antibody.

The use of the term “flat dose” with regard to the methods and dosagesdescribed herein means a dose that is administered to a patient withoutregard for the weight or body surface area (BSA) of the patient. Theflat dose is therefore not provided as a mg/kg dose, but rather as anabsolute amount of the agent (e.g., the anti-TIM3 antibody). Forexample, a 60 kg person and a 100 kg person would receive the same doseof an antibody (e.g., 480 mg of an anti-TIM3 antibody).

As used herein, the terms “ug” and “uM” are used interchangeably with“μg” and “μM,” respectively.

Various aspects described herein are described in further detail in thefollowing subsections.

I. Anti-Human TIM3 Antibodies

Described herein are antibodies, e.g., fully human antibodies, which arecharacterized by particular functional features or properties. Forexample, the antibodies specifically bind human TIM3, and morespecifically, a particular domain (e.g., a functional domain) within theextracellular domain of human TIM3. In a particular embodiment, theantibodies specifically bind to the site on TIM3 to which TIM3-L binds.In certain embodiments, the antibodies are antagonist antibodies, i.e.,they inhibit or suppress the T cell inhibitory activity of TIM3 oncells, e.g., T cells. In certain embodiments, anti-TIM3 antibodiescross-react with TIM3 from one or more non-human primates, such ascynomolgus TIM3. In certain embodiments, the antibodies specificallybind to the extracellular region of human TIM3 and the extracellularregion of cynomolgus TIM3. In one embodiment, the antibodies bind tohuman TIM3 with high affinity.

Anti-TIM3 antibodies described herein exhibit one or more of thefollowing functional properties:

(a) binding to soluble and/or membrane bound human TIM3;

(b) binding to soluble and/or membrane bound cyno TIM3;

(c) inducing or stimulating an immune response;

(d) inducing or stimulating T cell activation, e.g., Th1 cell activation(as evidenced, e.g., by enhanced cytokine secretion and/orproliferation);

(e) inducing or stimulating T cell proliferation (e.g., CD4+, CD8+ Tcells, Th1 cells, or TILs), e.g., in a coculture assay, such asdescribed in the Examples;

(f) inducing or stimulating IFN-γ production by T cells, e.g., Th1 cellsor tumor infiltrating lymphocytes (TILs), such as TILs from human renal,lung, pancreatic, or breast cancer tumors, as determined, e.g., in theassay described in the Examples;

(g) blocking or inhibiting the binding of human TIM3 to PtdSer, asdetermined, e.g., in the assay described in the Examples;

(h) not internalizing or downregulating cell surface TIM3 when bindingto TIM3 on cells;

(i) binding to human TIM3 extracellular domain (i) CPVFECG (SEQ ID NO:296); (ii) RIQIPGIMND (SEQ ID NO: 298); (iii) CPVFECG and RIQIPGIMND(SEQ ID NOs: 296 and 298, respectively); or (iv) WTSRYWLNGDFR (SEQ IDNO: 297);

(j) competing with, or cross-blocking, the binding to human TIM3 of anantibody binding to TIM3 described herein (e.g., 13A3, 3G4, 17C3, 17C8,9F6, or any of TIM3.2 to TIM3.18), as determined, e.g., in the assaydescribed in the Examples;

(k) binding to human TIM3, but not to human TIM3 having an amino acidsubstitution of one or more of the following amino acid residues: L48,C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87,R89, D104, R111, Q113, G116, M118, and D120, as numbered in SEQ ID NO:286 (FIG. 20); and(l) binding to human TIM3 regions ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367)and ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) as determined by HDX-MS;(m) having the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography; and/or(n) competing with or cross-blocking with the binding to human TIM3 of13A3 or TIM3.18.IgG1.3, e.g., as described in the Examples.

In some embodiments, anti-TIM3 antibodies described herein bind to humanTIM3 with high affinity, for example, with a K_(D) of 10⁻⁷ M or less,10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, 10⁻² Mor less, 10⁻² M to 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁷ M, or 10⁻⁹M to 10⁻⁷ M. In certain embodiments, an anti-TIM3 antibody binds tosoluble human TIM3, e.g., as determined by BIACORE™ (e.g., as describedin the Examples), with a K_(D) of 10⁻⁷M or less, 10⁻⁸ M or less, 10⁻⁹ M(1 nM) or less, 10⁻¹⁰ M or less, 10⁻² M to 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁷ M,10⁻¹ M to 10⁻⁷ M, 10⁻⁹M to 10⁻⁷ M, or 10⁻⁸M to 10⁻⁷ M. In certainembodiments, an anti-TIM3 antibody binds to bound (e.g., cell membranebound) human TIM3, such as on activated human T cells, e.g., asdetermined by flow cytometry and Scatchard plot, with a K_(D) of 10⁻⁷ Mor less, 10⁻⁸ M or less, 10⁻⁹ M (1 nM) or less, 5×10⁻¹⁰ M or less, 10⁻¹⁰M or less, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁸ M, 10⁻¹⁰M to 10⁻⁸ M, 10⁻⁹M to 10⁻⁸ M, 10⁻¹¹ M to 10⁻⁹ M, or 10⁻¹⁰ M to 10⁻⁹ M. In certainembodiments, an anti-TIM3 antibody binds to bound (e.g., cell membranebound) human TIM3, such as on activated human T cells, e.g., asdetermined by flow cytometry, with an EC₅₀ of 10 ug/mL or less, 5 ug/mLor less, 1 ug/mL or less, 0.9 ug/mL or less, 0.8 ug/mL or less, 0.7ug/mL or less, 0.6 ug/mL or less, 0.5 ug/mL or less, 0.4 ug/mL or less,0.3 ug/mL or less, 0.2 ug/mL or less, 0.1 ug/mL or less, 0.05 ug/mL orless, or 0.01 ug/mL or less. In some embodiments, anti-TIM3 antibodiesdescribed herein bind to cyno TIM3, for example, with a K_(D) of 10⁻⁷ Mor less, 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ M orless, 10⁻¹² M or less, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁷ M, 10⁻¹⁰ M to10⁻⁷ M, or 10⁻⁹ M to 10⁻⁷ M. In certain embodiments, an anti-TIM3antibody binds to soluble cyno TIM3, e.g., as determined by BIACORE™(e.g., as described in the Examples), with a K_(D) of 10⁻⁷ M or less,10⁻⁸ M or less, 10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹² M to 10⁻⁷M, 10⁻¹¹ M to 10⁻⁷ M, 10⁻¹⁰M to 10⁻⁷ M, 10⁻⁹M to 10⁻⁷ M, or 10⁻⁸ M to10⁻⁷ M. Anti-TIM3 antibodies can bind to membrane bound cynomolgus TIM3,e.g., with an EC₅₀ of 100 nM or less, 10 nM or less, 100 nM to 0.01 nM,100 nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1 nM, e.g., as measured byflow cytometry (e.g., as described in the Examples). In certainembodiments, an anti-TIM3 antibody binds to bound (e.g., cell membranebound) cyno TIM3, such as on activated human T cells, e.g., asdetermined by flow cytometry and Scatchard plot, with a K_(D) of 10⁻⁷ Mor less, 10⁻⁸ M or less, 10⁻⁹ M (1 nM) or less, 5×10⁻¹⁰ M or less, 10⁻¹⁰M or less, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁸ M, 10⁻¹⁰ M to 10⁻⁸ M, 10⁻⁹M to 10⁻⁸ M, 10⁻¹¹ M to 10⁻⁹ M, or 10⁻¹⁰ M to 10⁻⁹ M.

In certain embodiments, anti-TIM3 antibodies described herein stimulateor enhance an immune response, e.g., by activating T cells, e.g., in thetumor. For example, the anti-TIM3 antibodies can activate or costimulatecells, as evidenced, e.g., by enhanced cytokine (e.g., IFN-γ) secretionand/or enhanced proliferation, which may result from the inhibition ofTIM3 mediated T cell inhibitory activity. In certain embodiments, T cellactivation or co-stimulation by a TIM3 antibody occurs in the presenceof CD3 stimulation. In certain embodiments, an anti-TIM3 antibodyincreases IFN-γ secretion by a factor of 50%, 100% (i.e., 2 fold), 3fold, 4 fold, 5 fold or more, optionally with a maximum of up to 10fold, 30 fold, 100 fold, as measured, e.g., on primary human T cellsand/or T cells expressing human TIM3, such as tumor infiltratinglymphocytes (TILs).

In certain embodiments, anti-TIM3 antibodies inhibit binding ofphosphatidylserine to human TIM3 on cells, e.g., CHO cells or activatedT cells expressing human TIM3, e.g., with an EC₅₀ of 10 μg/ml or less, 1μg/ml or less, 0.01 μg/ml to 10 μg/ml, 0.1 μg/ml to 10 μg/ml, or 0.1μg/ml to 1 μg/ml.

In certain embodiments, anti-TIM3 antibodies described herein bind to anepitope, e.g., a conformational epitope, in the extracellular portion ofhuman TIM3, e.g., in the Ig like domain of the extracellular region,i.e., amino acids 22 to 202 of SEQ ID NO: 286 (FIG. 20). In certainembodiments, an anti-TIM3 antibody binds to an epitope located withinamino acids 22 to 120 of human TIM3 extracellular domain (SEQ ID NO:286) or 1-99 of mature human TIM3 (SEQ ID NO: 290) (see Examples). Incertain embodiments, an anti-TIM3 antibody binds to, or to an epitopewithin, a region consisting of amino acids 58-64 of human TIM3 havingSEQ ID NO: 286, which corresponds to amino acid residues 37-43 of maturehuman TIM3 (CPVFECG, SEQ ID NO: 296; see FIG. 20). In certainembodiments, an anti-TIM3 antibody binds to, or to an epitope within, aregion consisting of amino acids 111-120 of human TIM3 having SEQ ID NO:286, which corresponds to amino acid residues 90-99 of mature human TIM3(RIQIPGIMND, SEQ ID NO: 298; see FIG. 20). In certain embodiments, ananti-TIM3 antibody binds to, or to an epitope within, a regionconsisting of a region consisting of amino acids 58-64 of human TIM3having SEQ ID NO: 286 (CPVFECG, SEQ ID NO: 296) and to, or to an epitopewithin, a region consisting of amino acids 111-120 of human TIM3 havingSEQ ID NO: 286 (RIQIPGIMND, SEQ ID NO: 298; see FIG. 20). In certainembodiments, an anti-TIM3 antibody binds to, or to an epitope within, aregion consisting of amino acids 78-89 of human TIM3 having SEQ ID NO:286, which corresponds to amino acid residues 57-83 of mature human TIM3(WTSRYWLNGDFR, SEQ ID NO: 297; see FIG. 20).

In one embodiment, an anti-TIM3 antibody binds to substantially the sameepitope as that of 13A3, i.e., an epitope (or region of human TIM3)comprising one or more of amino acid residues C58, P59, F61, E62, C63,R111, and D120 of SEQ ID NO: 286 (FIG. 20). In some embodiments, ananti-TIM3 antibody binds to an epitope (or region of human TIM3)comprising one or more of amino acid residues C58, P59, F61, E62, C63,D104, R111, Q113 and D120 of SEQ ID NO: 286 (FIG. 20). In certainembodiments, an anti-TIM3 antibody does not bind significantly, or onlywith significantly reduced binding affinity, to a human TIM3 protein inwhich one or more of amino acid residues C58, P59, F61, E62, C63, R111,and D120 of SEQ ID NO: 286 is changed to another amino acid, e.g., in anon-conservative amino acid substitution. In certain embodiments, ananti-TIM3 antibody does not bind significantly, or only withsignificantly reduced binding affinity, to a human TIM3 protein in whichone or more of amino acid residues C58, P59, F61, E62, C63, D104, R111,Q113 and D120 of SEQ ID NO: 286 is changed to another amino acid, e.g.,in a non-conservative amino acid substitution.

In some embodiments, an anti-TIM3 antibody binds to substantially thesame epitope as that of 3G4, i.e., an epitope (or region of human TIM3)comprising one or more of amino acids residues C58, P59, V60, F61, E62,C63, G116, and M118 of SEQ ID NO: 286 (FIG. 20). In some embodiments, ananti-TIM3 antibody binds to an epitope (or region of human TIM3)comprising one or more of amino acid residues C58, P59, V60, F61, E62,C63, D104, G116, and M118 of SEQ ID NO: 286 (FIG. 20). In certainembodiments, an anti-TIM3 antibody does not bind significantly, or onlywith significantly reduced binding affinity, to a human TIM3 protein inwhich one or more of amino acid residues C58, P59, V60, F61, E62, C63,G116, and M118 of SEQ ID NO: 286 is changed to another amino acid, e.g.,in a non-conservative amino acid substitution. In certain embodiments,an anti-TIM3 antibody does not bind significantly, or only withsignificantly reduced binding affinity, to a human TIM3 protein in whichone or more of amino acid residues C58, P59, V60, F61, E62, C63, D104,G116, and M118 of SEQ ID NO: 286 is changed to another amino acid, e.g.,in a non-conservative amino acid substitution.

In some embodiments, an anti-TIM3 antibody binds to substantially thesame epitope as that of 17C3, i.e., an epitope (or region of human TIM3)comprising one or more of amino acids residues C58, P59, V60, F61, E62,C63, G64, and G116 of SEQ ID NO: 286 (FIG. 20). In some embodiments, ananti-TIM3 antibody binds to an epitope (or region of human TIM3)comprising one or more of amino acid residues C58, P59, V60, F61, E62,C63, G64, D104, and G116 of SEQ ID NO: 286 (FIG. 20). In certainembodiments, an anti-TIM3 antibody does not bind significantly, or onlywith significantly reduced binding affinity, to a human TIM3 protein inwhich one or more of amino acid residues C58, P59, V60, F61, E62, C63,G64, and G116 of SEQ ID NO: 286 is changed to another amino acid, e.g.,in a non-conservative amino acid substitution. In certain embodiments,an anti-TIM3 antibody does not bind significantly, or only withsignificantly reduced binding affinity, to a human TIM3 protein in whichone or more of amino acid residues C58, P59, V60, F61, E62, C63, G64,D104, and G116 of SEQ ID NO: 286 is changed to another amino acid, e.g.,in a non-conservative amino acid substitution.

In some embodiments, an anti-TIM3 antibody binds to substantially thesame epitope as that of 8B9, i.e., an epitope (or region of human TIM3)comprising one or more of amino acids residues L48, W78, S80, R81, W83,G86, D87, and R89 of SEQ ID NO: 286 (FIG. 20). In some embodiments, ananti-TIM3 antibody binds to an epitope (or region of human TIM3)comprising one or more of amino acid residues L48, W78, S80, R81, W83,L84, G86, D87, and R89 of SEQ ID NO: 286 (FIG. 20). In some embodiments,an anti-TIM3 antibody binds to substantially the same epitope as that of8B9, i.e., an epitope (or region of human TIM3) comprising one or moreof amino acids residues L48, W78, S80, R81, W83, G86, D87, R89, and D104of SEQ ID NO: 286 (FIG. 20). In certain embodiments, an anti-TIM3antibody does not bind significantly, or only with significantly reducedbinding affinity, to a human TIM3 protein in which one or more of aminoacid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO:286 (FIG. 20) is changed to another amino acid, e.g., in anon-conservative amino acid substitution. In certain embodiments, ananti-TIM3 antibody does not bind significantly, or only withsignificantly reduced binding affinity, to a human TIM3 protein in whichone or more of amino acid residues L48, W78, S80, R81, W83, L84, G86,D87, and R89 of SEQ ID NO: 286 (FIG. 20) is changed to another aminoacid, e.g., in a non-conservative amino acid substitution. In someembodiments an anti-TIM3 antibody does not bind significantly, or onlywith significantly reduced binding affinity, to a human TIM3 protein inwhich one or more of amino acid residues L48, W78, S80, R81, W83, G86,D87, R89, and D104 of SEQ ID NO: 286 (FIG. 20) is changed to anotheramino acid, e.g., in a non-conservative amino acid substitution.

In certain embodiments, anti-TIM3 antibodies compete for binding tohuman TIM3 with (or inhibit binding of) anti-TIM3 antibodies comprisingCDRs or variable regions described herein, e.g., those of antibodies13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 and any of TIM3.2 to TIM3.18. Incertain embodiments, anti-TIM3 antibodies inhibit binding of antibodies13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any of TIM3.2 to TIM3.18 tohuman TIM3 by at least 50%, 60%, 70%, 80%, 90% or by 100%. In certainembodiments, 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any of TIM3.2 toTIM3.18 inhibit binding of anti-TIM3 antibodies to human TIM3 by atleast 50%, 60%, 70%, 80%, 90% or by 100%. In certain embodiments,anti-TIM3 antibodies inhibit binding of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9,8C4 or any of TIM3.2 to TIM3.18 to human TIM3 by at least 50%, 60%, 70%,80%, 90% or by 100% and 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any ofTIM3.2 to TIM3.18 inhibit binding of the anti-TIM3 antibodies to humanTIM3 by at least 50%, 60%, 70%, 80%, 90% or by 100% (e.g., compete inboth directions).

In certain embodiments, anti-TIM3 antibodies have 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or all of the following features:

(1) binding to soluble human TIM3, e.g., with a K_(D) of 10 nM or less(e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;

(2) binding to soluble cynomolgus TIM3, e.g., with a K_(D) of 100 nM orless (e.g., 0.01 nM to 100 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;

(3) binding to membrane bound human TIM3, e.g., with an EC₅₀ of 1 ug/mLor less (e.g., 0.01 ug/mL to 1 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);

(4) binding to membrane bound human TIM3, e.g., with a K_(D) of 1 nM orless (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard analysis,e.g., as described in the Examples;

(5) binding to membrane bound cynomolgus TIM3, e.g., with an EC₅₀ of 20ug/mL or less (e.g., 0.01 ug/mL to 20 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);

(6) binding to membrane bound cynomolgus TIM3, e.g., with a K_(D) of 1nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchardanalysis, e.g., as described in the Examples;

(7) inducing or enhancing T cell activation (e.g., by blocking orreducing the inhibitory effects of TIM3), as evidenced by (i) increasedIFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs)and/or (ii) enhanced proliferation of TIM-3 expressing T cells (e.g.,Th1 cells or TILs), e.g., as described in the Examples;(8) stimulating T cell proliferation in a mixed lymphocyte reaction(MLR) assay, e.g., as described in the Examples;(9) inhibiting the binding of phosphatidylserine to TIM3, e.g., asmeasured by PS-hTIM3 “in-tandem” blocking assay, e.g., as described inthe Examples;(10) not internalizing or downregulating cell surface TIM3 when bindingto TIM3 on cells;(11) binding to one of the following regions of human TIM3 extracellulardomain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND(SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298,respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297), e.g., as describedin the Examples;(12) having reduced binding to human TIM3 in which one or more of aminoacids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84,G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (as numbered inSEQ ID NO: 286 (FIG. 20)) is substituted with another amino acidrelative to binding to wildtype human TIM3, e.g., as described in theExamples;(13) competing in either direction or both directions for binding tohuman TIM3 with an antibody comprising VH and VL domains of any one of13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or TIM3.7, TIM3.8, TIM3.10,TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.18, e.g., asdescribed in the Examples;(14) binding to human TIM3 regions ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367)and ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) as determined by HDX-MS,e.g., as described in the Examples;(15) having the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography (e.g., describedin the Examples; numbering per SEQ ID NO: 286 (FIG. 20)); and/or(16) (a) having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6,7, 8 or 9 of amino acids C58, P59, F61, E62, C63, R111, D120, andoptionally D104 and Q113 (numbering per SEQ ID NO: 286 (FIG. 20)) aresubstituted with another amino acid relative to binding to wildtypehuman TIM3; (b) binding to ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367),¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) and ¹¹⁹NDEKFNLKL¹²⁷ (SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and/or (c)competing with or cross-blocking with the binding to human TIM3 of 13A3or TIM3.18.IgG1.3, e.g., as described in the Examples.

Accordingly, an antibody that exhibits one or more of these functionalproperties (e.g., biochemical, immunochemical, cellular, physiologicalor other biological activities, or the like) as determined according tomethodologies known to the art and described herein, will be understoodto exhibit a statistically significant difference in the particularactivity relative to that seen in the absence of the antibody (e.g., orwhen a control antibody of irrelevant specificity is present). In someembodiments, anti-TIM3 antibody-induced increases in a measuredparameter (e.g., T cell proliferation, cytokine production) in a givenassay effects a statistically significant increase by at least 10% ofthe measured parameter, e.g., by at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 100% (i.e., 2 fold), 3 fold, 5 fold or 10 fold, and incertain embodiments, an antibody described herein can increase themeasured parameter, e.g., by greater than 92%, 94%, 95%, 97%, 98%, 99%,100% (i.e, 2 fold), 3 fold, 5 fold or 10 fold, relative to the sameassay conducted in the absence of the antibody. Conversely, anti-TIM3antibody-induced decreases in a measured parameter (e.g., tumor volume,TIM3-L binding to human TIM3) in a given assay effects a statisticallysignificant decrease by at least 10% of the measured parameter, e.g., byat least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and in certainembodiments, an antibody described herein can decrease the measuredparameter, e.g., by greater than 92%, 94%, 95%, 97%, 98% or 99%,relative to the same assay conducted in the absence of the antibody.

Standard assays to evaluate the binding ability of the antibodies towardTIM3 of various species are known in the art, including for example,ELISAs, Western blots, and RIAs. Suitable assays are described in detailin the Examples. The binding kinetics (e.g., binding affinity) of theantibodies can also be assessed by standard assays known in the art,such as by Biacore analysis. Assays to evaluate the effects of theantibodies on functional properties of TIM3 (e.g., ligand binding, Tcell proliferation, cytokine production) are described in further detailinfra and in the Examples.

In certain embodiments, anti-TIM3 antibodies are not native antibodiesor are not naturally-occurring antibodies. For example, anti-TIM3antibodies have post-translational modifications that are different fromthose of antibodies that are naturally occurring, such as by havingmore, less or a different type of post-translational modification.

In certain embodiments, anti-TIM3 antibodies do not have agonistactivity, as determined, e.g., in cross-linking of anti-TIM3 antibodiesin CHO-OKT3-CD32:T cell co-culture experiments, in which such antibodiesdo not enhance activity beyond anti-TIM3 alone. In certain embodiments,anti-TIM3 antibodies block the interaction of TIM3 with its ligandwithout promoting agonist activity.

In certain embodiments, anti-TIM3 antibodies enhance IL-12 productionfrom monocytes or dendritic cells treated with LPS.

In certain embodiments, anti-TIM3 antibodies revive tumor infiltratingCD8+ T cells that coexpress PD-1 and TIM3 by combined treatment, henceavoiding depletion of CD8+ T cells.

II. Exemplary Anti-TIM3 Antibodies

Particular anti-TIM3 antibodies described herein are antibodies, e.g.,monoclonal, recombinant, and/or human antibodies, having the CDR and/orvariable region sequences of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9,8C4 or any one of TIM3.2 to TIM3.18, isolated and structurallycharacterized as described herein, as well as antibodies having at least80% identity (e.g., at least 85%, at least 90%, at least 95%, or atleast 99% identity) to their variable region or CDR sequences. The VHamino acid sequences of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, and 17C8 are setforth in SEQ ID NOs: 34-40, respectively. The VH amino acid sequences ofmutated versions of 13A3, 8B9 and 9F6 are set forth in SEQ ID NOs:112-121, and 364. The VL amino acid sequences of 13A3, 17C3, and 3G4 areset forth in SEQ ID NO: 60. The VL amino acid sequences of 8B9, 8C4, and17C8 are set forth in SEQ ID NO: 61. The VL amino acid sequence of 9F6are set forth in SEQ ID NOs: 61, 62, and 63. The VL amino acid sequencesof the mutated versions of 13A3, 8B9 and 9F6 are those of thecorresponding nonmutated antibodies. A summary of the identity of SEQ IDNOs is provided in FIG. 13.

Accordingly, provided herein are isolated antibodies, or antigen bindingportion thereof, comprising heavy and light chain variable regions,wherein the heavy chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 34-40, 112-121 and364.

Also provided are isolated antibodies, or antigen binding portionsthereof, comprising heavy and light chain variable regions, wherein thelight chain variable region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 60-63.

Provided herein are isolated anti-human TIM3 antibodies, orantigen-binding portion thereof, comprising:

(a) heavy and light chain variable region sequences comprising SEQ IDNOs: 34 and 60, respectively;

(b) heavy and light chain variable region sequences comprising SEQ IDNOs: 35 and 61, respectively;

(c) heavy and light chain variable region sequences comprising SEQ IDNOs: 36 and 61, respectively;

(d) heavy and light chain variable region sequences comprising SEQ IDNOs: 37 and 60, respectively;

(e) heavy and light chain variable region sequences comprising SEQ IDNOs: 38 and 61, respectively;

(f) heavy and light chain variable region sequences comprising SEQ IDNOs: 38 and 62, respectively;

(g) heavy and light chain variable region sequences comprising SEQ IDNOs: 38 and 63, respectively;

(h) heavy and light chain variable region sequences comprising SEQ IDNOs: 39 and 60, respectively;

(i) heavy and light chain variable region sequences comprising SEQ IDNOs: 40 and 61, respectively;

(j) heavy and light chain variable region sequences comprising SEQ IDNOs: 121 and 63, respectively;

(k) heavy and light chain variable region sequences comprising SEQ IDNOs: 120 and 61, respectively;

(l) heavy and light chain variable region sequences comprising SEQ IDNOs: 112 and 60, respectively;

(m) heavy and light chain variable region sequences comprising SEQ IDNOs: 113 and 60, respectively;

(n) heavy and light chain variable region sequences comprising SEQ IDNOs: 114 and 60, respectively;

(o) heavy and light chain variable region sequences comprising SEQ IDNOs: 115 and 60, respectively;

(p) heavy and light chain variable region sequences comprising SEQ IDNOs: 116 and 60, respectively;

(q) heavy and light chain variable region sequences comprising SEQ IDNOs: 117 and 60, respectively;

(r) heavy and light chain variable region sequences comprising SEQ IDNOs: 118 and 60, respectively;

(s) heavy and light chain variable region sequences comprising SEQ IDNOs: 119 and 60, respectively; or

(t) heavy and light chain variable region sequences comprising SEQ IDNOs: 364 and 60, respectively.

Anti-TIM3 antibodies can comprise the heavy and light chain CDR1s, CDR2sand CDR3s of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, and 17C8 or any one ofTIM3.2 to TIM3.18, or combinations thereof. The amino acid sequences ofthe VH CDR1s of 13A3, 8B9, 8C4, and 17C3 are set forth in SEQ ID NOs:41-44, respectively. The amino acid sequences of the VH CDR1s of 9F6,3G4, and 17C8 are set forth in SEQ ID NO 45. The amino acid sequence ofthe VH CDR1 of the mutated 13A3 antibodies (i.e., TIM3.10-TIM3.18) isthe same as that of the nonmutated 13A3 antibody, i.e., SEQ ID NO: 41.The amino acid sequence of the VH CDR1 of the mutated 8B9 antibody(i.e., TIM3.8) is the same as that of the nonmutated 8B9 antibody, i.e.,SEQ ID NO: 42. The amino acid sequence of the VH CDR1 of the mutated 9F6antibody (i.e., TIM3.7) is the same as that of the nonmutated 9F6antibody, i.e., SEQ ID NO: 45. The amino acid sequences of the VH CDR2sof 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, and 17C8 are set forth in SEQ ID NOs:46-52, respectively. The amino acid sequence of the VH CDR2s of themutated 13A3 antibodies TIM3.10, TIM3.17, and TIM3.18 is set forth inSEQ ID NO: 122. The amino acid sequence of the VH CDR2s of the mutated13A3 antibodies TIM3.11 and TIM3.12 are set forth in SEQ ID NOs: 123 and124, respectively. The amino acid sequence of the VH CDR2 of the mutated13A3 antibodies TIM3.13 and TIM3.16 is that of the nonmutated 13A3antibody, i.e., SEQ ID NO: 46. The amino acid sequence of the VH CDR2 ofthe mutated 8B9 antibody (i.e., TIM3.8) is set forth in SEQ ID NO: 125.The amino acid sequence of the VH CDR2 of the mutated 9F6 antibody(i.e., TIM3.7) is the same as that of the nonmutated 9F6 antibody, i.e.,SEQ ID NO: 45. The amino acid sequences of the VH CDR3s of 13A3, 8B9,8C4, 17C3, 9F6, 3G4, and 17C8 are set forth in SEQ ID NOs: 53-59,respectively.

The amino acid sequence of the VH CDR3s of the mutated 13A3 antibodies(i.e., TIM3.10 to TIM3.12 is that of the nonmutated 13A3 antibody, i.e.,SEQ ID NO: 53. The amino acid sequence of the VH CDR3s of the mutated13A3 antibodies TIM3.13 and TIM3.18 is set forth in SEQ ID NO: 126. Theamino acid sequence of the VH CDR3s of the mutated 13A3 antibodiesTIM3.15 and TIM3.17 is set forth in SEQ ID NO: 128. The amino acidsequences of the VH CDR3s of the mutated 13A3 antibodies TIM3.14 andTIM3.16 are set forth in SEQ ID NOs: 127 and 129, respectively. Theamino acid sequence of the VH CDR3 of the mutated 8B9 antibody (i.e.,TIM3.8) is that of the nonmutated 8B9 antibody, i.e., SEQ ID NO: 54. Theamino acid sequence of the VH CDR3 of the mutated 9F6 antibody (i.e.,TIM3.7) is the same as that of the nonmutated 9F6 antibody, i.e., SEQ IDNO: 57. The amino acid sequences of the VH CDR3s of 13A3, 8B9, 8C4,17C3, 9F6, 3G4, and 17C8 are set forth in SEQ ID NOs: 53-59,respectively. The amino acid sequences of the VL CDR1s of 13A3, 8B9,8C4, 17C3, 3G4, and 17C8 are set forth in SEQ ID NO: 64. The amino acidsequences of the VL CDR1 of 9F6 is set forth in SEQ ID NOs: 64 and 65.The amino acid sequences of the VL CDR2s of 13A3, 8B9, 8C4, 17C3, 3G4,and 17C8 are set forth in SEQ ID NO: 66. The amino acid sequences of theVL CDR2 of 9F6 is set forth in SEQ ID NOs: 66 and 67. The amino acidsequences of the VL CDR3s of 13A3, 17C3, and 3G4 are set forth in SEQ IDNO: 68. The amino acid sequences of the VL CDR3s of 8B9, 8C4, and 17C8are set forth in SEQ ID NO: 69. The amino acid sequences of the VL CDR3of 9F6 are set forth in SEQ ID NOs: 69, 70, and 71. The amino acidsequences of the VL CDRs of the mutated antibodies 13A3, 8B9 and 9F6 arethose of the corresponding nonmutated antibodies. FIG. 13 provides alist of the SEQ ID NOs for the CDRs of anti-TIM3 antibodies describedherein.

The CDR regions are delineated using the Kabat system (Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242). Kabat system is the most common numbering system for ascheme called the EU index or EU numbering system, which is based on thesequential numbering of the first human IgG1 sequenced (the EU antibody;Edelman et al. 1969). Based on the Kabat numbering scheme disclosedherein, the antibody numbering can be converted into other systems knownin the art, e.g., Chotia, IMGT, Martin (enhanced Chothia), or AHonumbering scheme.

Given that each of these antibodies bind to human TIM3 and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequences,e.g., those in FIG. 13, can be “mixed and matched” (i.e., CDRs fromdifferent antibodies can be mixed and match, although each antibody mustcontain a VH CDR1, 2 and 3 and a VL CDR1, 2 and 3) to create otheranti-TIM3 binding molecules described herein. TIM3 binding of such“mixed and matched” antibodies can be tested using the binding assaysdescribed above and in the Examples (e.g., ELISAs). In some embodiments,when VH CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3sequence from a particular VH sequence is replaced with a structurallysimilar CDR sequence(s). Likewise, when VL CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular VLsequence is replaced with a structurally similar CDR sequence(s). Itwill be readily apparent to the ordinarily skilled artisan that novel VHand VL sequences can be created by substituting one or more VH and/or VLCDR region sequences with structurally similar sequences from the CDRsequences disclosed herein for monoclonal antibodies 13A3, 8B9, 8C4,17C3, 9F6, 3G4, 17C8 and any one of TIM3.2 to TIM3.18.

Provided herein are isolated anti-human TIM3 antibodies, or antigenbinding portion thereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 41-45;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 46-52 and 122-125;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 53-59 and 126-129;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 64-65;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 66-67; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 68-71; wherein theantibody specifically binds to human TIM3.

In one embodiment, the anti-human TIM3 antibody comprises heavy andlight chain variable regions, wherein the heavy chain variable regionCDR1, CDR2, and CDR3 regions comprise:

(a) SEQ ID NOs: 41, 46, 53;

(b) SEQ ID NOs: 42, 47, 54;

(c) SEQ ID NOs: 43, 48, 55;

(d) SEQ ID NOs: 44, 49, 56;

(e) SEQ ID NOs: 45, 50, 57;

(f) SEQ ID NOs: 45, 51, 58;

(g) SEQ ID NOs: 45, 52, 59;

(h) SEQ ID NOs: 41, 122, 53;

(i) SEQ ID NOs: 41, 123, 53;

(j) SEQ ID NOs: 41, 124, 53;

(k) SEQ ID NOs: 41, 46, 126;

(l) SEQ ID NOs: 41, 46, 127;

(m) SEQ ID NOs: 41, 46, 128;

(n) SEQ ID NOs: 41, 46, 129;

(o) SEQ ID NOs: 41, 122, 128; or

(p) SEQ ID NOs: 41, 122, 126; wherein the antibody specifically binds tohuman TIM3.

In some embodiments, the anti-human TIM3 antibody comprises heavy andlight chain variable regions, wherein the light chain variable regionCDR1, CDR2, and CDR3 regions comprise:

(a) SEQ ID NOs: 64, 66, 68;

(b) SEQ ID NOs: 64, 66, 69;

(c) SEQ ID NOs: 65, 67, 70; or

(d) SEQ ID NOs: 64, 66, 71; wherein the antibody specifically binds tohuman TIM3.

In a particular embodiment, the anti-TIM3 antibody comprises heavy andlight chain variable regions, wherein:

(a1) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a2) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 122, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a3) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 123, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a4) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 124, 53, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a5) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 126, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a6) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 127, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a7) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 128, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a8) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 46, 129, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a9) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 122, 128, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(a10) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 41, 122, 126, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(b1) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 42, 47, 54, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;

(b2) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 42, 125, 54, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;

(c) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 43, 48, 55, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;

(d) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 44, 49, 56, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively;

(e) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 45, 50, 57, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;

(f) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 45, 50, 57, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 71, respectively;

(g1) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 45, 50, 57, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 65, 67, 70, respectively;

(g2) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 45, 50, 57, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 71, respectively;

(g3) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 45, 50, 57, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;

(h) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 45, 51, 58, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 68, respectively; or(i) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 45, 52, 59, respectively, and the light chain variable regionCDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 66, 69, respectively;wherein the antibody specifically binds to human TIM3.

A VH domain, or one or more CDRs thereof, described herein can be linkedto a constant domain for forming a heavy chain, e.g., a full lengthheavy chain. Similarly, a VL domain, or one or more CDRs thereof,described herein can be linked to a constant domain for forming a lightchain, e.g., a full length light chain. A full length heavy chain (withthe exception of the C-terminal lysine (K) or with the exception of theC-terminal glycine and lysine (GK), which can be absent) and full lengthlight chain combine to form a full length antibody.

A VH domain described herein can be fused to the constant domain of ahuman IgG, e.g., IgG1, IgG2, IgG3 or IgG4, which are eithernaturally-occurring or modified, e.g., as further described herein. Forexample, a VH domain can comprise the amino acid sequence of any VHdomain described herein fused to a human IgG, e.g., an IgG1, constantregion, such as the following wild-type human IgG1 constant domain aminoacid sequence:

(SEQ ID NO: 291) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKor that of an allotypic variant of SEQ ID NO: 291 and have the followingamino acid sequences:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 277;allotype specific amino acid residues are in bold and underlined)

A VH domain of an anti-TIM3 antibody can comprise the amino acidsequence of any VH domain described herein fused to an effectorlessconstant region, e.g., the following effectorless human IgG1 constantdomain amino acid sequences:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 294; “IgG1.1f,”comprising substitutions L234A, L235E, G237A, A330S and P331S, which areunderlined) or ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E E M TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 295;“IgG1.3f”, comprising substitutions L234A,L235E and G237A, which are underlined)

For example, an allotypic variant of IgG1 comprises an K97R, D239E,and/or L241M (underlined and bolded above) and numbering according tothat in SEQ ID NOs: 277, 294, and 295. Within the full length heavyregion, e.g., 8C4 (SEQ ID NO: 3) and according to EU numbering, theseamino acid substitutions are numbered K214R, D356E, and L358M. In someembodiments, the constant region of an anti-TIM3 antibody can furthercomprises one or more mutations or substitutions at amino acids L117,A118, G120, A213, and P214 (underlined above) as numbered in SEQ ID NO:277, 294, and 295, or L234, A235, G237, A330 and P331, per EU numbering.In further embodiments, the constant region of an anti-TIM3 antibodycomprises one or more mutations or substitutions at amino acids L117A,A118E, G120A, A213S, and P214S of SEQ ID NO: 291, or L234A, L235E,G237A, A330S and P331S, per EU numbering. The constant region of ananti-TIM3 antibody may also comprise one or more mutations orsubstitutions L117A, A118E and G120A of SEQ ID NO: 291, or L234A, L235Eand G237A, per EU numbering

Alternatively, a VH domain of an anti-TIM3 antibody can comprise theamino acid sequence of any VH domain described herein fused to a humanIgG4 constant region, e.g., the following human IgG4 amino acid sequenceor variants thereof:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. (SEQ ID NO: 292, comprising S228P)

A VL domain described herein can be fused to the constant domain of ahuman Kappa or Lambda light chain. For example, a VL domain of ananti-TIM3 antibody can comprise the amino acid sequence of any VL domaindescribed herein fused to the following human IgG1 kappa light chainamino acid sequence:

(SEQ ID NO: 278) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

In certain embodiments, the heavy chain constant region comprises alysine or another amino acid at the C-terminus, e.g., it comprises thefollowing last amino acids: LSPGK (SEQ ID NO: 279) in the heavy chain.In certain embodiments, the heavy chain constant region is lacking oneor more amino acids at the C-terminus, and has, e.g., the C-terminalsequence LSPG (SEQ ID NO: 280) or LSP (SEQ ID NO: 281).

The amino acid sequences of exemplary heavy and light chains correspondto SEQ ID NOs: 1-28, 72-111, 301-354, for the heavy chains and SEQ IDNOs: 29-30 and 32-33 for the light chains.

Provided herein are isolated anti-human TIM3 antibodies, orantigen-binding portion thereof, comprising:

(a1) heavy and light chain sequences comprising SEQ ID NOs: 301 (or 302)and 29, respectively;

(a2) heavy and light chain sequences comprising SEQ ID NOs: 1 (or 8) and29, respectively;

(a3) heavy and light chain sequences comprising SEQ ID NOs: 15 (or 22)and 29, respectively;

(a4) heavy and light chain sequences comprising SEQ ID NOs: 303 (or 304)and 29, respectively;

(a5) heavy and light chain sequences comprising SEQ ID NOs: 72 (or 82)and 29, respectively;

(a6) heavy and light chain sequences comprising SEQ ID NOs: 92 (or 102)and 29, respectively;

(a7) heavy and light chain sequences comprising SEQ ID NOs: 305 (or 306)and 29, respectively;

(a8) heavy and light chain sequences comprising SEQ ID NOs: 73 (or 83)and 29, respectively;

(a9) heavy and light chain sequences comprising SEQ ID NOs: 93 (or 103)and 29, respectively;

(a10) heavy and light chain sequences comprising SEQ ID NOs: 307 (or308) and 29, respectively;

(a11) heavy and light chain sequences comprising SEQ ID NOs: 74 (or 84)and 29, respectively;

(a12) heavy and light chain sequences comprising SEQ ID NOs: 94 (or 104)and 29, respectively;

(a13) heavy and light chain sequences comprising SEQ ID NOs: 309 (or310) and 29, respectively;

(a14) heavy and light chain sequences comprising SEQ ID NOs: 75 (or 85)and 29, respectively;

(a15) heavy and light chain sequences comprising SEQ ID NOs: 95 (or 105)and 29, respectively;

(a16) heavy and light chain sequences comprising SEQ ID NOs: 311 (or312) and 29, respectively;

(a17) heavy and light chain sequences comprising SEQ ID NOs: 76 (or 86)and 29, respectively;

(a18) heavy and light chain sequences comprising SEQ ID NOs: 96 (or 106)and 29, respectively;

(a19) heavy and light chain sequences comprising SEQ ID NOs: 313 (or314) and 29, respectively;

(a20) heavy and light chain sequences comprising SEQ ID NOs: 77 (or 87)and 29, respectively;

(a21) heavy and light chain sequences comprising SEQ ID NOs: 97 (or 107)and 29, respectively;

(a22) heavy and light chain sequences comprising SEQ ID NOs: 315 (or316) and 29, respectively;

(a23) heavy and light chain sequences comprising SEQ ID NOs: 78 (or 88)and 29, respectively;

(a24) heavy and light chain sequences comprising SEQ ID NOs: 98 (or 108)and 29, respectively;

(a25) heavy and light chain sequences comprising SEQ ID NOs: 317 (or318) and 29, respectively;

(a26) heavy and light chain sequences comprising SEQ ID NOs: 79 (or 89)and 29, respectively;

(a27) heavy and light chain sequences comprising SEQ ID NOs: 99 (or 109)and 29, respectively;

(a28) heavy and light chain sequences comprising SEQ ID NOs: 319 (or320) and 29, respectively;

(a29) heavy and light chain sequences comprising SEQ ID NOs: 349 (or350) and 29, respectively;

(a30) heavy and light chain sequences comprising SEQ ID NOs: 351 (or352) and 29, respectively;

(a31) heavy and light chain sequences comprising SEQ ID NOs: 353 (or354) and 29, respectively;

(b1) heavy and light chain sequences comprising SEQ ID NOs: 321 (or 322)and 30, respectively;

(b2) heavy and light chain sequences comprising SEQ ID NOs: 2 (or 9) and30, respectively;

(b3) heavy and light chain sequences comprising SEQ ID NOs: 16 (or 23)and 30, respectively;

(b4) heavy and light chain sequences comprising SEQ ID NOs: 323 (or 324)and 30, respectively;

(b5) heavy and light chain sequences comprising SEQ ID NOs: 80 (or 90)and 30, respectively;

(b6) heavy and light chain sequences comprising SEQ ID NOs: 100 (or 110)and 30, respectively;

(b7) heavy and light chain sequences comprising SEQ ID NOs: 325 (or 326)and 30, respectively;

(c1) heavy and light chain sequences comprising SEQ ID NOs: 327 (or 328)and 30, respectively;

(c2) heavy and light chain sequences comprising SEQ ID NOs: 3 (or 10)and 30, respectively;

(c3) heavy and light chain sequences comprising SEQ ID NOs: 17 (or 24)and 30, respectively;

(c4) heavy and light chain sequences comprising SEQ ID NOs: 329 (or 330)and 30, respectively;

(d1) heavy and light chain sequences comprising SEQ ID NOs: 331 (or 332)and 29, respectively;

(d2) heavy and light chain sequences comprising SEQ ID NOs: 4 (or 11)and 29, respectively;

(d3) heavy and light chain sequences comprising SEQ ID NOs: 18 (or 25)and 29, respectively;

(d4) heavy and light chain sequences comprising SEQ ID NOs: 333 (or 334)and 29, respectively;

(e1.1) heavy and light chain sequences comprising SEQ ID NOs: 335 (or336) and 32, respectively;

(e1.2) heavy and light chain sequences comprising SEQ ID NOs: 335 (or336) and 33, respectively;

(e1.3) heavy and light chain sequences comprising SEQ ID NOs: 335 (or336) and 31, respectively;

(e2) heavy and light chain sequences comprising SEQ ID NOs: 5 (or 12)and 33, respectively;

(e3) heavy and light chain sequences comprising SEQ ID NOs: 19 (or 26)and 33, respectively;

(e4) heavy and light chain sequences comprising SEQ ID NOs: 337 (or 338)and 33, respectively;

(e5) heavy and light chain sequences comprising SEQ ID NOs: 81 (or 91)and 33, respectively;

(e6) heavy and light chain sequences comprising SEQ ID NOs: 101 (or 111)and 33, respectively;

(e7) heavy and light chain sequences comprising SEQ ID NOs: 339 (or 340)and 33, respectively;

(f1) heavy and light chain sequences comprising SEQ ID NOs: 341 (or 342)and 29, respectively;

(f2) heavy and light chain sequences comprising SEQ ID NOs: 6 (or 13)and 29, respectively;

(f3) heavy and light chain sequences comprising SEQ ID NOs: 20 (or 27)and 29, respectively;

(f4) heavy and light chain sequences comprising SEQ ID NOs: 343 (or 344)and 29, respectively;

(g1) heavy and light chain sequences comprising SEQ ID NOs: 345 (or 346)and 30, respectively;

(g2) heavy and light chain sequences comprising SEQ ID NOs: 7 (or 14)and 30, respectively;

(g3) heavy and light chain sequences comprising SEQ ID NOs: 21 (or 28)and 30, respectively; or

(g4) heavy and light chain sequences comprising SEQ ID NOs: 347 (or 348)and 30, respectively; wherein the antibody specifically binds to humanTIM3.

In certain embodiments, an anti-TIM3 antibody comprises a combination ofa heavy and light chain sequences set forth herein, e.g., in thepreceding paragraph, wherein the antibody comprises two heavy chains andtwo light chains, and can further comprise at least one disulfide bondlinking the two heavy chains together. The antibodies can also comprisedisulfide bonds linking each of the light chains to each of the heavychains.

Heavy and light chains comprising an amino acid sequence that is atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or 70% identical toany of the heavy or light chains set forth herein (or their variableregions), e.g., SEQ ID NOs: 1-33, 72-111, and 301-354 can be used forforming anti-human TIM3 antibodies having the desired characteristics,e.g., those further described herein. Exemplary variants are thosecomprising an allotypic variation, e.g., in the constant domain, and/ora mutation in the variable or constant region, such as the mutationsdisclosed herein. Heavy and light chains comprising an amino acidsequence that differs in at most 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4,1-3, 1-2 or 1 amino acid (by substitution, addition or deletion) fromany of the heavy or light chains set forth herein (or their variableregions) can be used for forming anti-human TIM3 antibodies having thedesired characteristics, e.g., those further described herein.

In various embodiments, the antibodies described above exhibit one ormore, two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten or more, eleven ormore, twelve or more, thirteen or more, fourteen or more, fifteen ormore, or all of the following functional properties:

(1) binding to soluble human TIM3, e.g., with a KD of 10 nM or less(e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;

(2) binding to soluble cynomolgus TIM3, e.g., with a KD of 100 nM orless (e.g., 0.01 nM to 100 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;

(3) binding to membrane bound human TIM3, e.g., with an EC50 of 1 ug/mLor less (e.g., 0.01 ug/mL to 1 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);

(4) binding to membrane bound human TIM3, e.g., with a K_(D) of 1 nM orless (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard analysis,e.g., as described in the Examples;

(5) binding to membrane bound cynomolgus TIM3, e.g., with an EC50 of 20ug/mL or less (e.g., 0.01 ug/mL to 20 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);

(6) binding to membrane bound cynomolgus TIM3, e.g., with a K_(D) of 1nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchardanalysis, e.g., as described in the Examples;

(7) inducing or enhancing T cell activation (e.g., by blocking orreducing the inhibitory effect of TIM3), as evidenced by (i) increasedIFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs)and/or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1cells or TILs), e.g., as described in the Examples;(8) stimulating T cell proliferation in a mixed lymphocyte reaction(MLR) assay, e.g., as described in the Examples;(9) inhibiting the binding of phosphatidylserine to TIM3, e.g., asmeasured by PS-hTIM3 “in-tandem” blocking assay, e.g., as described inthe Examples;(10) not internalizing or downregulating cell surface TIM3 when bindingto TIM3 on cells;(11) binding to one of the following regions of human TIM3 extracellulardomain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND(SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298,respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297), e.g., as describedin the Examples;(12) having reduced binding to human TIM3 in which one or more of aminoacids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84,G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (as numbered inSEQ ID NO: 286 (FIG. 20)) is substituted with another amino acidrelative to binding to wildtype human TIM3, e.g., as described in theExamples;(13) competing in either direction or both directions for binding tohuman TIM3 with an antibody comprising VH and VL domains of any one of13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or TIM3.7, TIM3.8, TIM3.10,TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, andTIM3.18, e.g., as described in the Examples;(14) binding to human TIM3 regions ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367)and ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) as determined by HDX-MS,e.g., as described in the Examples;(15) having the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography (e.g., describedin the Examples; numbering per SEQ ID NO: 286 (FIG. 20)); and/or(16) (a) having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6,7, 8 or 9 of amino acids C58, P59, F61, E62, C63, R111, D120, andoptionally D104 and Q113 (numbering per SEQ ID NO: 286 (FIG. 20)) aresubstituted with another amino acid relative to binding to wildtypehuman TIM3; (b) binding to ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367),¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) and ¹¹⁹NDEKFNLKL¹²⁷ (SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and/or (c)competing with or cross-blocking with the binding to human TIM3 of 13A3or TIM3.18.IgG1.3, e.g., as described in the Examples.

Such antibodies include, for example, human antibodies, humanizedantibodies, or chimeric antibodies.

In one embodiment, anti-TIM3 antibodies described herein bind to aconformational epitope.

In one embodiment, anti-TIM3 antibodies described herein bind to aminoacid residues within the following region of mature human TIM3extracellular domain (SEQ ID NO: 290):SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMND (SEQ ID NO: 299), corresponding to amino acidresidues 1-99 of mature human TIM3 extracellular domain (SEQ ID NO: 290)or amino acids 22 to 120 of human TIM3 having SEQ ID NO: 286.

In one embodiment, anti-TIM3 antibodies described herein bind to aminoacid residues within the following region of mature human TIM3extracellular domain (SEQ ID NO: 290): CPVFECG (SEQ ID NO: 296),corresponding to amino acid residues 37-43 of mature human TIM3extracellular domain (SEQ ID NO: 290).

In one embodiment, anti-TIM3 antibodies described herein bind to aminoacid residues within the following region of mature human TIM3extracellular domain (SEQ ID NO: 290): WTSRYWLNGDFR (SEQ ID NO: 297),corresponding to amino acid residues 57-83 of mature human TIM3extracellular domain (SEQ ID NO: 290).

In one embodiment, anti-TIM3 antibodies described herein bind to aminoacid residues within the following region of mature human TIM3extracellular domain (SEQ ID NO: 290): RIQIPGIMND (SEQ ID NO:298),corresponding to amino acid residues 90-99 of mature human TIM3extracellular domain (SEQ ID NO: 290).

In one embodiment, anti-TIM3 antibodies have the same pattern of bindingto wildtype and mutated human TIM3 as that of one or more of antibodies13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 and TIM3.2 to TIM3.18. In oneembodiment, an anti-TIM3 antibody binds to amino acid residues withinthe following regions of mature human TIM3 extracellular domain (SEQ IDNO: 290): CPVFECG (SEQ ID NO: 296), WTSRYWLNGDFRKGDVSLTIENVTLAD (SEQ IDNO: 297), and/or RIQIPGIMND (SEQ ID NO: 298).

In certain embodiments, an anti-TIM3 antibody binds to(1)⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367) and ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQID NO: 368) or (2) ⁴⁰YTPAAPGNLVPVCWGKGACPVFE⁶² (SEQ ID NO: 369),⁶⁶VVLRTDERDVNY⁷⁷ (SEQ ID NO: 370), ⁷⁸WTSRYWLNGDFRKGDVSL⁹⁵ (SEQ ID NO:371), ¹¹⁰CRIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 372), and ¹¹⁹NDEKFNLKL¹²⁷(SEQ ID NO: 373), as determined by HDX-MS, as described, e.g., in theExamples. In certain embodiments, an anti-TIM3 antibody interacts withregions of amino acid residues 40-62 and 111-127 of hTIM3, but does notsignificantly interact with other regions, such as the region that isN-terminal to amino acid residue Y40, the region that is located betweenamino acid residues E62 and R111, and the region that is C-terminal toamino acid residue L127, as determined by HDX-MS, as described, e.g., inthe Examples.

In certain embodiments, an anti-TIM3 antibody has reduced binding tohuman TIM3 in which one or more of amino acids L48, C58, P59, V60, F61,E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113,G116, M118, and D120 (as numbered in SEQ ID NO: 286 (FIG. 20)) issubstituted with another amino acid relative to binding to wildtypehuman TIM3 and the antibody binds to (1) ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO:367) and ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) or (2)⁴⁰YTPAAPGNLVPVCWGKGACPVFE⁶² (SEQ ID NO: 369), ⁶⁶VVLRTDERDVNY⁷⁷ (SEQ IDNO: 370), ⁷⁸WTSRYWLNGDFRKGDVSL⁹⁵ (SEQ ID NO: 371),¹¹⁰CRIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 372), and ¹¹⁹NDEKFNLKL¹²⁷ (SEQ IDNO: 373), as determined by HDX-MS, as described, e.g., in the Examples.

In certain embodiments, an anti-TIM3 antibody has a similar pattern ofbinding to wild-type and mutated human TIM3 as that of TIM3.18.IgG1.3 or13A3, i.e., the antibody:

(i) binds to (1)⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367),111RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368), and ¹¹⁹NDEKFNLKL¹²⁷ (SEQ IDNO: 373), and, e.g., but does not bind significantly to (a) peptideshaving sequences located N-terminal of amino acid residue 49; (b)peptides having sequences located between amino acid residue 62 and 111(e.g., ⁷⁸WTSRYWLNGDFRKGDVSL⁹⁵ (SEQ ID NO: 371)); and (c) peptides havingsequences that are located C-terminal of amino acid residue 127, asdetermined by HDX-MS (e.g., as described in the Examples);

(ii) fails to bind to human TIM3, or has significantly reduced bindingto human TIM3, having one or more of the following amino acid mutations,as determined, e.g., using a yeast surface display method (e.g., asdescribed in the Examples): C58, P59, F61, E62, C63, R111, D120, andoptionally D104 and Q113 (numbering per SEQ ID NO: 286 (FIG. 20));and/or

(iii) has the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography (e.g., asdescribed in the Examples; numbering per SEQ ID NO: 286 (FIG. 20)).

In certain embodiments, an anti-TIM3 antibody comprises a heavy chainand a light chain, wherein the heavy chain is selected from the groupconsisting of SEQ ID NOs: 72-111, 305-354, 325-326, and 339-340, and/orthe light chain is selected from the group consisting of SEQ ID NOs:29-33.

As further discussed herein, the heavy chain constant region ofanti-TIM3 antibodies described herein can be of any isotype, e.g., IgG1,IgG2, IgG3 and IgG4, or combinations thereof and/or modificationsthereof. An anti-TIM3 antibody can have effector function or can havereduced or no effector function. In certain embodiments, anti-TIM3antibodies comprise a modified heavy chain constant region that providesenhanced properties to the antibody.

In certain embodiments, an anti-TIM3 antibody comprises a heavy chainand an light chain, wherein the heavy chain is selected from the groupconsisting of SEQ ID NOs: 72-111, and 349-352, and/or the light chain isselected from the group consisting of SEQ ID NOs: 29-33.

III. Antibodies Having Particular Germline Sequences

In certain embodiments, an anti-TIM3 antibody comprises a heavy chainvariable region from a particular germline heavy chain immunoglobulingene and/or a light chain variable region from a particular germlinelight chain immunoglobulin gene.

As demonstrated herein, human antibodies specific for TIM3 have beenprepared that comprise a heavy chain variable region that is the productof or derived from a human germline VH 4-39 gene, VH 4-59 gene, VH 1-46gene, VH 3-11, VH 4-17 gene, VH 3-10 gene, VH 6-19 gene, VH 6-13 gene,VH JH5b gene and/or VH JH6b gene. Accordingly, provided herein areisolated monoclonal antibodies, or antigen-binding portions thereof,comprising a heavy chain variable region that is the product of orderived from a human VH germline gene selected from the group consistingof: VH 4-39, VH 4-59, VH 1-46, VH 3-11, VH 4-17, VH 3-10, VH6-19, VH6-13, VH JH5b, VH JH6b, and any combination thereof.

Human antibodies specific for TIM3 have been prepared that comprise alight chain variable region that is the product of or derived from ahuman germline VK A27 gene, VK JK5 gene, VK JK4 gene, VK L18 gene,and/or VK JK1 gene. Accordingly, provide herein are isolated monoclonalantibodies, or antigen-binding portions thereof, comprising a lightchain variable region that is the product of or derived from a human VKgermline gene selected from the group consisting of: VK A27, VK JK5, VKJK4, VK L18, VK JK1, and any combination thereof.

Anti-TIM3 antibodies described herein include those comprising a heavychain variable region that is the product of or derived from one of theabove-listed human germline VH genes and also comprising a light chainvariable region that is the product of or derived from one of theabove-listed human germline VK genes, as shown in the Figures.

As used herein, a human antibody comprises heavy and light chainvariable regions that are “the product of or “derived from” a particulargermline sequence if the variable regions of the antibody are obtainedfrom a system that uses human germline immunoglobulin genes. Suchsystems include immunizing a transgenic mouse carrying humanimmunoglobulin genes with the antigen of interest or screening a humanimmunoglobulin gene library displayed on phage with the antigen ofinterest. A human antibody that is “the product of or “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequences of human germline immunoglobulins and selecting the humangermline immunoglobulin sequence that is closest in sequence (i.e.,greatest % identity) to the sequence of the human antibody. A humanantibody that is “the product of or “derived from” a particular humangermline immunoglobulin sequence can contain amino acid differences ascompared to the germline sequence, due to, for example,naturally-occurring somatic mutations or intentional introduction ofsite-directed mutation. However, a selected human antibody typically isat least 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the human antibody as being human when comparedto the germline immunoglobulin amino acid sequences of other species(e.g., murine germline sequences). In certain cases, a human antibodycan be at least 95%, or even at least 96%, 97%, 98%, or 99% identical inamino acid sequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody candisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

IV. Homologous Antibodies

Encompassed herein are antibodies having heavy and light chain variableregions comprising amino acid sequences that are homologous to the aminoacid sequences of the anti-TIM3 antibodies described herein, and whereinthe antibodies retain the desired functional properties of the anti-TIM3antibodies described herein.

For example, an isolated anti-TIM3 antibody, or antigen binding portionthereof, can comprise a heavy chain variable region and a light chainvariable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:34-40, 112-121, and 364, or comprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5,1-10, 1-15, 1-20, 1-25, or 1-50 amino acid changes (i.e., amino acidsubstitutions, additions or deletions) relative to an amino acidsequence selected from the group consisting of SEQ ID NOs: 34-40,112-121, and 364, wherein optionally the heavy chain variable regioncomprises the CDR sequences of one of the anti-TIM3 antibodies describedherein;(b) the light chain variable region comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:60-63, or comprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20,1-25, or 1-50 amino acid changes (i.e., amino acid substitutions,additions or deletions) relative to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 60-63, wherein optionally the lightchain variable region comprises the CDR sequences of one of theanti-TIM3 antibodies described herein;(c) the antibody specifically binds to human TIM3, and(d) the antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or all of the following functional properties:(1) binding to soluble human TIM3, e.g., with a K_(D) of 10 nM or less(e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;(2) binding to soluble cynomolgus TIM3, e.g., with a K_(D) of 100 nM orless (e.g., 0.01 nM to 100 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;(3) binding to membrane bound human TIM3, e.g., with an EC₅₀ of 1 ug/mLor less (e.g., 0.01 ug/mL to 1 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);(4) binding to membrane bound human TIM3, e.g., with a K_(D) of 1 nM orless (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard analysis,e.g., as described in the Examples;(5) binding to membrane bound cynomolgus TIM3, e.g., with an EC₅₀ of 20ug/mL or less (e.g., 0.01 ug/mL to 20 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);(6) binding to membrane bound cynomolgus TIM3, e.g., with a K_(D) of 1nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchardanalysis, e.g., as described in the Examples;(7) inducing or enhancing T cell activation (e.g., by blocking orreducing the inhibitory effect of TIM3), as evidenced by (i) increasedIFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs)and/or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1cells or TILs), e.g., as described in the Examples;(8) stimulating T cell proliferation in a mixed lymphocyte reaction(MLR) assay, e.g., as described in the Examples;(9) inhibiting the binding of phosphatidylserine to TIM3, e.g., asmeasured by PS-hTIM3 “in-tandem” blocking assay, e.g., as described inthe Examples;(10) not internalizing or downregulating cell surface TIM3 when bindingto TIM3 on cells;(11) binding to one of the following regions of human TIM3 extracellulardomain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND(SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298,respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297), e.g., as describedin the Examples;(12) having reduced binding to human TIM3 in which one or more of aminoacids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84,G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (as numbered inSEQ ID NO: 286 (FIG. 20)) is substituted with another amino acidrelative to binding to wildtype human TIM3, e.g., as described in theExamples;(13) competing in either direction or both directions for binding tohuman TIM3 with an antibody comprising VH and VL domains of any one of13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or TIM3.7, TIM3.8, TIM3.10,TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, andTIM3.18, e.g., as described in the Examples;(14) binding to human TIM3 regions ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367)and ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) as determined by HDX-MS,e.g., as described in the Examples;(15) having the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography (e.g., describedin the Examples; numbering per SEQ ID NO: 286 (FIG. 20)); and/or(16) (a) having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6,7, 8 or 9 of amino acids C58, P59, F61, E62, C63, R111, D120, andoptionally D104 and Q113 (numbering per SEQ ID NO: 286 (FIG. 20)) aresubstituted with another amino acid relative to binding to wildtypehuman TIM3; (b) binding to ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367),¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) and ¹¹⁹NDEKFNLKL¹²⁷ (SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and/or (c)competing with or cross-blocking with the binding to human TIM3 of 13A3or TIM3.18.IgG1.3, e.g., as described in the Examples.

In various embodiments, the antibody can exhibit one or more, two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, or all of the functional propertieslisted as (1) through (16) above. The antibody can be, for example, ahuman antibody, a humanized antibody or a chimeric antibody.

An isolated anti-TIM3 antibody, or antigen binding portion thereof, cancomprise a heavy chain and a light chain, wherein:

(a) the heavy chain comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-28, 72-111,and 349-352, or comprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15,1-20, 1-25, or 1-50 amino acid changes (i.e., amino acid substitutions,additions or deletions) relative to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1-28, 72-111, and 349-352, with theproviso that, in certain embodiments, if the sequence is that of aneffectorless heavy chain, the mutations rendering the heavy chaineffectorless are not modified (e.g., no modification is made to R214,A234, E235, A237, S330 and S331) for IgG1.1 constant regions, and nomodification is made to R214, A234 and E235 for IgG1.3 constant regions,wherein optionally the heavy chain variable region comprises the CDRsequences of one of the anti-TIM3 antibodies described herein;(b) the light chain comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 29-31, orcomprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or1-50 amino acid changes (i.e., amino acid substitutions, additions ordeletions) relative to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 29-31, wherein optionally the light chainvariable region comprises the CDR sequences of one of the anti-TIM3antibodies described herein;(c) the antibody specifically binds to human TIM3, and (d) the antibodyexhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all ofthe following functional properties:(1) binding to soluble human TIM3, e.g., with a K_(D) of 10 nM or less(e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;(2) binding to soluble cynomolgus TIM3, e.g., with a K_(D) of 100 nM orless (e.g., 0.01 nM to 100 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;(3) binding to membrane bound human TIM3, e.g., with an EC₅₀ of 1 ug/mLor less (e.g., 0.01 ug/mL to 1 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);(4) binding to membrane bound human TIM3, e.g., with a K_(D) of 1 nM orless (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard analysis,e.g., as described in the Examples;5) binding to membrane bound cynomolgus TIM3, e.g., with an EC₅₀ of 20ug/mL or less (e.g., 0.01 ug/mL to 20 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);(6) binding to membrane bound cynomolgus TIM3, e.g., with a K_(D) of 1nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchardanalysis, e.g., as described in the Examples;(7) inducing or enhancing T cell activation (e.g., by blocking orreducing the inhibitory effect of TIM3), as evidenced by (i) increasedIFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs)and/or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1cells or TILs), e.g., as described in the Examples;(8) stimulating T cell proliferation in a mixed lymphocyte reaction(MLR) assay, e.g., as described in the Examples;(9) inhibiting the binding of phosphatidylserine to TIM3, e.g., asmeasured by PS-hTIM3 “in-tandem” blocking assay, e.g., as described inthe Examples;(10) not internalizing or downregulating cell surface TIM3 when bindingto TIM3 on cells;(11) binding to one of the following regions of human TIM3 extracellulardomain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND(SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298,respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297), e.g., as describedin the Examples;(12) having reduced binding to human TIM3 in which one or more of aminoacids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84,G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (as numbered inSEQ ID NO: 286 (FIG. 20)) is substituted with another amino acidrelative to binding to wildtype human TIM3, e.g., as described in theExamples;(13) competing in either direction or both directions for binding tohuman TIM3 with an antibody comprising VH and VL domains of any one of13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, TIM3.7, TIM3.8, TIM3.10, TIM3.11,TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, and TIM3.18, e.g.,as described in the Examples;(14) binding to human TIM3 regions ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367)and ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) as determined by HDX-MS,e.g., as described in the Examples;(15) having the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography (e.g., describedin the Examples; numbering per SEQ ID NO: 286 (FIG. 20)); and/or(16) (a) having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6,7, 8 or 9 of amino acids C58, P59, F61, E62, C63, R111, D120, andoptionally D104 and Q113 (numbering per SEQ ID NO: 286 (FIG. 20)) aresubstituted with another amino acid relative to binding to wildtypehuman TIM3; (b) binding to ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367),¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) and ¹¹⁹NDEKFNLKL¹²⁷ (SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and/or (c)completing with or cross-blocking with the binding to human TIM3 of 13A3or TIM3.18.IgG1.3, e.g., as described in the Examples.

Also provided are anti-TIM3 antibodies comprising a VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and/or VL CDR3 that differs from thecorresponding CDR of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any ofTIM3.2 to TIM3.18 in 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 amino acidchanges (i.e., amino acid substitutions, additions or deletions). Incertain embodiments, an anti-TIM3 antibody comprises 1-5 amino acidchanges in each of 1, 2, 3, 4, 5 or 6 of the CDRs relative to thecorresponding sequence in 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any ofTIM3.2 to TIM3.18. In certain embodiments, an anti-TIM3 antibodycomprises at total of 1-5 amino acid changes across all CDRs relative tothe CDRs in 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any of TIM3.2 toTIM3.18.

In certain embodiments, an anti-TIM3 antibody comprises VH and VL CDRsconsisting of those of 13A3, wherein one or more of the amino acids inone or more CDRs are those of one of the other anti-TIM3 antibodiesdisclosed herein.

For example, in certain embodiments, an anti-TIM3 antibody comprises aVH CDR1 comprising one or more amino acid modifications relative toSRSYYWG (SEQ ID NO: 41), and can comprise, e.g., the followingdegenerate sequence: X₁X₂X₃X₄YX₅X₆(SEQ ID NO: 282), wherein X₁ is anyamino acid, e.g., S or none; X₂ is any amino acid, e.g., R or none; X₃is any amino acid, e.g., S, R, or D; X₄ is any amino acid, e.g., Y or H;X₅ is any amino acid, e.g., W or M; and X₆ is any amino acid, e.g., G,N, S, or H.

In certain embodiments, an anti-TIM3 antibody comprises a VH CDR2comprising one or more amino acid modifications relative toSIYYSGFTYYNPSLKS (SEQ ID NO: 46), and can comprise, e.g., the followingdegenerate sequence: X₁IX₂X₃X₄GX₅X₆X₇X₈YX₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO:283), wherein X₁ is any amino acid, e.g., S, Y, I, or F; X₂ is any aminoacid, e.g., Y, H, N, or S; X₃ is any amino acid, e.g., Y, P, G, T, or S;X₄ is any amino acid, e.g., S, T, R, or G; X₅ is any amino acid, e.g.,F, S, or D; X₆ is any amino acid, e.g., S, T, or I; X₇ is any aminoacid, e.g., I or none; X₈ is any amino acid, e.g., Y, N, or I; X₉ is anyamino acid, e.g., N, Q, S, or A; X₁₀ is any amino acid, e.g., P, S, Q,or D; X₁₁ is any amino acid, e.g., S or K; X₁₂ is any amino acid, e.g.,L, F, or V; X₁₃ is any amino acid, e.g., K or Q; and X₁₄ is any aminoacid, e.g., S or G.

In certain embodiments, an anti-TIM3 antibody comprises a VH CDR3comprising one or more amino acid modifications relative toGGPYGDYAHWFDP (SEQ ID NO: 53), and can comprise, e.g., the followingdegenerate sequence: X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀YGX₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈(SEQ ID NO: 284), wherein X₁ is any amino acid, e.g., D, E, or none; X₂is any amino acid, e.g., F, G, or none; X₃ is any amino acid, e.g., Y ornone; X₄ is any amino acid, e.g., G, S, or none; X₅ is any amino acid,e.g., G, T, or S; X₆ is any amino acid, e.g., G or S; X₇ is any aminoacid, e.g., N, W, or none; X₈ is any amino acid, e.g., Y, S, E, or none;X₉ is any amino acid, e.g., Y or none; X₁₀ is any amino acid, e.g., P orY; X₁₁ is any amino acid, e.g., D or none; X₁₂ is any amino acid, e.g.,Y or none; X₁₃ is any amino acid, e.g., A or none; X₁₄ is any aminoacid, e.g., H or none; X₁₅ is any amino acid, e.g., W or none; X₁₆ isany amino acid, e.g., F or M; X₁₇ is any amino acid, e.g., D or E; andX₁₈ is any amino acid, e.g., P, I, V, Y, or L.

In certain embodiments, an anti-TIM3 antibody comprises a VH CDR3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 53-59 and 126-129.

In certain embodiments, an anti-TIM3 antibody comprises a VL CDR1comprising an amino acid sequence as set forth in SEQ ID NO: 64 or SEQID NO: 65.

In certain embodiments, an anti-TIM3 antibody comprises a VL CDR2comprising an amino acid sequence as set forth in SEQ ID NO: 66 or SEQID NO: 67.

In certain embodiments, an anti-TIM3 antibody comprises a VL CDR3comprising one or more amino acid modifications relative to QQYGSSPIT(SEQ ID NO: 68), and can comprise, e.g., the following degeneratesequence: QQX₁X₂SX₃X₄X₅T (SEQ ID NO: 285), wherein X₁ is any amino acid,e.g., F or Y; X₂ is any amino acid, e.g., N or G; X₃ is any amino acid,e.g., Y or S; X₄ is any amino acid, e.g., P or none; X₅ is any aminoacid, e.g., I, R, or L.

Antibodies having sequences with homology to those of 13A3, 8B9, 8C4,17C3, 9F6, 3G4, 8C4 or any of TIM3.2 to TIM3.18, e.g., the VH and VLregions of SEQ ID NOs: 34-40, 112-121, or 364, and SEQ ID NOs: 60-63,respectively, or heavy and light chains of SEQ ID NOs: 1-28, 72-111, or349-352, and SEQ ID NOs: 29-33, respectively, or CDRs, can be obtainedby mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) ofnucleic acid molecules encoding SEQ ID NOs: 167-173 and/or SEQ ID NOs:193-196 or SEQ ID NOs: 134-161 and/or SEQ ID NOs: 162-166, followed bytesting of the encoded altered antibody for retained function (i.e., thefunctions set forth in (1) through (16) above) using the functionalassays described herein.

V. Antibodies with Conservative Modifications

Anti-TIM3 antibodies can comprise a heavy chain variable regioncomprising CDR1, CDR2 and CDR3 sequences and a light chain variableregion comprising CDR1, CDR2 and CDR3 sequences, wherein one or more ofthese CDR sequences comprise specified amino acid sequences based on theanti-TIM3 antibodies described herein (e.g., 13A3, 3G4, 17C3, 17C8, 9F6,8B9, 8C4 or any of TIM3.2 to TIM3.18), or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the anti-TIM3 antibodies described herein. Accordingly, anisolated anti-TIM3 antibody, or antigen binding portion thereof, cancomprise a heavy chain variable region comprising CDR1, CDR2, and CDR3sequences and a light chain variable region comprising CDR1, CDR2, andCDR3 sequences, wherein:

(a) the heavy chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs: 53-59 and 126-129, and conservative modificationsthereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative aminoacid substitutions, wherein optionally the heavy chain variable regioncomprises the CDR sequences of one of the anti-TIM3 antibodies describedherein;(b) the light chain variable region CDR3 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequenceof SEQ ID NOs: 68-71, and conservative modifications thereof, e.g., 1,2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions,wherein optionally the light chain variable region comprises the CDRsequences of one of the anti-TIM3 antibodies described herein;(c) the antibody specifically binds to human TIM3, and (d) the antibodyexhibits 1, 2, 3, 4, 5, 6, 7, 8, 9 or all of the following features:(1) binding to soluble human TIM3, e.g., with a K_(D) of 10 nM or less(e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;(2) binding to soluble cynomolgus TIM3, e.g., with a K_(D) of 100 nM orless (e.g., 0.01 nM to 100 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;(3) binding to membrane bound human TIM3, e.g., with an EC₅₀ of 1 ug/mLor less (e.g., 0.01 ug/mL to 1 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);(4) binding to membrane bound human TIM3, e.g., with a K_(D) of 1 nM orless (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard analysis,e.g., as described in the Examples;(5) binding to membrane bound cynomolgus TIM3, e.g., with an EC₅₀ of 20ug/mL or less (e.g., 0.01 ug/mL to 20 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);(6) binding to membrane bound cynomolgus TIM3, e.g., with a K_(D) of 1nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchardanalysis, e.g., as described in the Examples;(7) inducing or enhancing T cell activation (e.g., by blocking orreducing the inhibitory effect of TIM3), as evidenced by (i) increasedIFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs)and/or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1cells or TILs), e.g., as described in the Examples;(8) stimulating T cell proliferation in a mixed lymphocyte reaction(MLR) assay, e.g., as described in the Examples;(9) inhibiting the binding of phosphatidylserine to TIM3, e.g., asmeasured by PS-hTIM3 “in-tandem” blocking assay, e.g., as described inthe Examples;(10) not internalizing or downregulating cell surface TIM3 when bindingto TIM3 on cells;(11) binding to one of the following regions of human TIM3 extracellulardomain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND(SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298,respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297), e.g., as describedin the Examples;(12) having reduced binding to human TIM3 in which one or more of aminoacids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84,G86, D87, R89, D104, R111, Q113, G116, M118 and D120 (as numbered in SEQID NO: 286 (FIG. 20)) is substituted with another amino acid relative tobinding to wildtype human TIM3, e.g., as described in the Examples;(13) competing in either direction or both directions for binding tohuman TIM3 with an antibody comprising VH and VL domains of any one of13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or TIM3.7, TIM3.8, TIM3.10,TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, andTIM3.18, e.g., as described in the Examples;(14) binding to human TIM3 regions ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367)and ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) as determined by HDX-MS,e.g., as described in the Examples;(15) having the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography (e.g., describedin the Examples; numbering per SEQ ID NO: 286 (FIG. 20)); and/or(16) (a) having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6,7, 8 or 9 of amino acids C58, P59, F61, E62, C63, R111, D120, andoptionally D104 and Q113 (numbering per SEQ ID NO: 286 (FIG. 20)) aresubstituted with another amino acid relative to binding to wildtypehuman TIM3; (b) binding to ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367),¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) and ¹¹⁹NDEKFNLKL¹²⁷ (SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and/or (c)competing with or cross-blocking with the binding to human TIM3 of 13A3or TIM3.18.IgG1.3, e.g., as described in the Examples.

In one embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 46-52 and 122-125, and conservativemodifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5conservative amino acid substitutions; and the light chain variableregion CDR2 sequence comprises an amino acid sequence selected from thegroup consisting of amino acid sequences of SEQ ID NOs: 66-67, andconservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4or 1-5 conservative amino acid substitutions.

In another embodiment, the heavy chain variable region CDR1 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 41-45, and conservativemodifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5conservative amino acid substitutions; and the light chain variableregion CDR1 sequence comprises an amino acid sequence selected from thegroup consisting of amino acid sequences of SEQ ID NOs: 64-65, andconservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4or 1-5 conservative amino acid substitutions.

In various embodiments, the antibody can exhibit one or more, two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, or all of the functional propertieslisted as (1) through (16) above. Such antibodies can be, for example,human antibodies, humanized antibodies or chimeric antibodies.

Conservative amino acid substitutions can also be made in portions ofthe antibodies other than, or in addition to, the CDRs. For example,conservative amino acid modifications can be made in a framework regionor in the Fc region. A variable region or a heavy or light chain cancomprise 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or1-50 conservative amino acid substitutions relative to the anti-TIM3antibody sequences provided herein. In certain embodiments, an anti-TIM3antibody comprises a combination of conservative and non-conservativeamino acid modification.

VI. Antibodies Binding to the Same Epitope or Competing for Binding

Also provided are antibodies that compete for binding to human TIM3 withone or more of the particular anti-TIM3 antibodies described herein(e.g., antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 and/orTIM3.2-TIM3.18). Such competing antibodies can be identified based ontheir ability to competitively inhibit binding to human TIM3 of one ormore of monoclonal antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4and/or TIM3.2-TIM3.18 in standard TIM3 binding assays. For example,standard ELISA assays or competitive ELISA assays can be used in which arecombinant human TIM3 protein is immobilized on the plate, variousconcentrations of unlabeled first antibody is added, the plate iswashed, labeled second antibody is added, and the amount of label ismeasured. If the increasing concentration of the unlabeled (first)antibody (also referred to as the “blocking antibody”) inhibits thebinding of the labeled (second) antibody, the first antibody is said toinhibit the binding of the second antibody to the target on the plate,or is said to compete with the binding of the second antibody.Additionally or alternatively, Biacore analysis can be used to assessthe ability of the antibodies to compete. The ability of a test antibodyto inhibit the binding of an anti-TIM3 antibody described herein to TIM3demonstrates that the test antibody can compete with the antibody forbinding to human TIM3.

Accordingly, provided herein are anti-TIM3 antibodies that inhibit thebinding of an anti-TIM3 antibodies described herein to TIM3 on cells,e.g., activated T cells, by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% and/orwhose binding to human TIM3 on cells, e.g., activated T cells, isinhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, e.g., as measured byELISA or FACS, such as by using the assay described in the followingparagraph.

An exemplary competition experiment to determine, e.g., whether a firstantibody blocks the binding of (i.e., “competes with”) a secondantibody, can be conducted as described in the Examples, or as follows:activated human T cells are prepared as follows: Peripheral BloodMononuclear Cells (PBMCs) are isolated from human whole blood usingFicoll gradient and activated with 10 μg/mL phytohaemagglutinin (PHA-L)(USBiol#P3370-30) and 200 IU/mL recombinant IL-2 (Peprotech#200-02) for3 days. The activated T cells are resuspended in FACS buffer (PBS with5% Fetal Bovine Serum) and seeded at 10⁵ cells per sample well in a 96well plate. The plate is set on ice followed by the addition ofunconjugated first antibody at concentrations ranging from 0 to 50 μg/mL(three-fold titration starting from a highest concentration of 50μg/mL). An unrelated IgG can be used as an isotype control for the firstantibody and added at the same concentrations (three-fold titrationstarting from a highest concentration of 50 μg/mL). A samplepre-incubated with 50 μg/mL unlabeled second antibody can be included asa positive control for complete blocking (100% inhibition) and a samplewithout antibody in the primary incubation can be used as a negativecontrol (no competition; 0% inhibition). After 30 minutes of incubation,labeled, e.g., biotinylated, second antibody is added at a concentrationof 2 μg/mL per well without washing. Samples are incubated for another30 minutes on ice. Unbound antibodies are removed by washing the cellswith FACS buffer. Cell-bound labeled second antibody is detected with anagent that detects the label, e.g., PE conjugated streptavidin(Invitrogen, catalog#S21388) for detecting biotin. The samples areacquired on a FACS Calibur Flow Cytometer (BD, San Jose) and analyzedwith FLOWJO® software (Tree Star, Inc, Ashland, Oreg.). The results canbe represented as the % inhibition (i.e., subtracting from 100% theamount of label at each concentration divided by the amount of labelobtained with no blocking antibody). Typically, the same experiment isthen conducted in the reverse, i.e., the first antibody is the secondantibody and the second antibody is the first antibody.

In certain embodiments, an antibody at least partially (e.g., at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or completely (100%)blocks the binding of the other antibody to the target, e.g., human TIM3or portion thereof, and regardless of whether inhibition occurs when oneor the other antibody is the first antibody. A first and a secondantibody “cross-block” binding of each other to the target, when theantibodies compete with each other both ways, i.e., in competitionexperiments in which the first antibody is added first and incompetition experiments in which the second antibody is added first.

In certain embodiments, anti-TIM3 antibodies bind to the same epitope asthat of the anti-TIM3 antibodies described herein (e.g., 13A3, 3G4,17C3, 17C8, 9F6, 8B9, 8C4 and/or TIM3.2-TIM3.18), e.g., as determined bya given epitope mapping technique. Techniques for determining antibodiesthat bind to the “same epitope on TIM3” with the anti-TIM3 antibodiesdescribed herein include, for example, epitope mapping methods, such as,x-ray analyses of crystals of antigen:antibody complexes which providesatomic resolution of the epitope. Other methods monitor the binding ofthe antibody to antigen fragments or mutated variations of the antigenwhere loss of binding due to a modification of an amino acid residuewithin the antigen sequence is often considered an indication of anepitope component (see FIG. 20). In addition, computationalcombinatorial methods for epitope mapping can also be used. Methods canalso rely on the ability of an antibody of interest to affinity isolatespecific short peptides (either in native three dimensional form or indenatured form) from combinatorial phage display peptide libraries. Thepeptides are then regarded as leads for the definition of the epitopecorresponding to the antibody used to screen the peptide library. Forepitope mapping, computational algorithms have also been developed whichhave been shown to map conformational discontinuous epitopes.

Antibodies that compete for binding with, or bind to the same epitopeas, the anti-TIM3 antibodies described herein can be identified by usingart-known methods. For example, mice can be immunized with human TIM3 asdescribed herein, hybridomas produced, and the resulting monoclonalantibodies screened for the ability to compete with an antibodydescribed herein for binding to human TIM3. Mice can also be immunizedwith a smaller fragment of TIM3 containing the epitope to which theantibody binds. The epitope or region comprising the epitope can belocalized by, e.g., screening for binding to a series of overlappingpeptides spanning TIM3. Alternatively, the method of Jespers et al.,Biotechnology 12:899, 1994 can be used to guide the selection ofantibodies having the same epitope and therefore similar properties toan anti-TIM3 antibody described herein. Using phage display, first theheavy chain of the anti-TIM3 antibody is paired with a repertoire of(human) light chains to select a TIM3-binding antibody, and then the newlight chain is paired with a repertoire of (human) heavy chains toselect a (human) TIM3-binding antibody having the same epitope orepitope region as an anti-TIM3 antibody described herein. Alternativelyvariants of an antibody described herein can be obtained by mutagenesisof cDNA encoding the heavy and light chains of the antibody.

Alanine scanning mutagenesis, as described by Cunningham and Wells(1989) Science 244: 1081-1085, or some other form of point mutagenesisof amino acid residues in TIM3 can also be used to obtain TIM3 antibodybinding characteristics.

Binding characteristics of a specific antibody can also be determined byassessing binding of the antibody to peptides comprising fragments ofTIM3, e.g., non-denatured or denatured fragments. A series ofoverlapping peptides encompassing the sequence of TIM3 (e.g., humanTIM3) can be synthesized and screened for binding, e.g., in a directELISA, a competitive ELISA (where the peptide is assessed for itsability to prevent binding of an antibody to TIM3 bound to a well of amicro titer plate), or on a chip.

Binding characteristics of anti-TIM3 antibodies can also be obtained byMS-based protein footprinting, such as Hydrogen/deuterium exchange massspectrometry (HDX-MS) and Fast Photochemical Oxidation of Proteins(FPOP). HDX-MS can be conducted, e.g., as described in WO2015/18735 andin Wei et al. (2014) Drug Discovery Today 19:95, the methods of whichare specifically incorporated by reference herein. FPOP can be conductedas described, e.g., in Hambley and Gross (2005) J. American Soc. MassSpectrometry 16:2057, the methods of which are specifically incorporatedby reference herein.

Binding characteristics anti-TIM3 antibodies can also be obtained bystructural methods, such as X-ray crystal structure determination (e.g.,WO2005/044853), molecular modeling and nuclear magnetic resonance (NMR)spectroscopy, including NMR determination of the H-D exchange rates oflabile amide hydrogens in TIM3 when free and when bound in a complexwith an antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31,11335-11347; Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

With regard to X-ray crystallography, crystallization can beaccomplished using any of the known methods in the art (e.g., Giege etal. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J.Biochem. 189: 1-23), including microbatch (e.g., Chayen (1997) Structure5: 1269-1274), hanging-drop vapor diffusion (e.g., McPherson (1976) J.Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to usea protein preparation having a concentration of at least about 1 mg/mLor about 10 mg/mL to about 20 mg/mL. Crystallization can be bestachieved in a precipitant solution containing polyethylene glycol1000-20,000 (PEG; average molecular weight ranging from about 1000 toabout 20,000 Da), about 5000 to about 7000 Da, or about 6000 Da, withconcentrations ranging from about 10% to about 30% (w/v). It can also bedesirable to include a protein stabilizing agent, e.g., glycerol at aconcentration ranging from about 0.5% to about 20%. A suitable salt,such as sodium chloride, lithium chloride or sodium citrate can also bedesirable in the precipitant solution, in a concentration ranging fromabout 1 mM to about 1000 mM. The precipitant is buffered to a pH of fromabout 3.0 to about 5.0. Specific buffers useful in the precipitantsolution can vary and are well-known in the art (Scopes, ProteinPurification: Principles and Practice, Third ed., (1994)Springer-Verlag, New York). Examples of useful buffers include, but arenot limited to, HEPES, Tris, MES and acetate. Crystals can be grow at awide range of temperatures, including 2° C., 4° C., 8° C. and 26° C.

Antibody: antigen crystals can be studied using well-known X-raydiffraction techniques and can be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g., Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H.W. Wyckoff et al., eds., Academic Press; U.S. Patent ApplicationPublication No. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst.D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet,eds.; Roversi et al. (2000) Acta Cryst. D56: 1313-1323), the disclosuresof which are hereby incorporated by reference in their entireties.

Anti-TIM3 antibodies can bind to the same epitope as any of theanti-TIM3 antibodies having amino acid sequences described herein, asdetermined by an epitope mapping technique, such as a techniquedescribed herein.

Antibodies binding to human TIM3 and optionally cyno TIM3 with similarbinding characteristics as the anti-TIM3 antibodies described herein anddetermined by one of the methods used in the Examples, are encompassedherein.

In certain embodiments, anti-TIM3 antibodies described herein bind to anepitope, e.g., a conformational epitope, in the extracellular portion ofhuman TIM3, e.g., in the Ig like domain or IgV domain of theextracellular region, i.e., amino acids 22 to 130 of SEQ ID NO: 286(FIG. 20). In certain embodiments, an anti-TIM3 antibody binds to anepitope located within amino acids 22 to 120 of human TIM3 extracellulardomain (SEQ ID NO: 286) or 1-99 of mature human TIM3 (SEQ ID NO: 290)(see Examples). In certain embodiments, an anti-TIM3 antibody binds to,or to an epitope within, a region consisting of amino acids 58-64 ofhuman TIM3 having SEQ ID NO: 286, which corresponds to amino acidresidues 37-43 of mature human TIM3 (CPVFECG, SEQ ID NO: 296; see FIG.20). In certain embodiments, an anti-TIM3 antibody binds to, or to anepitope within, a region consisting of amino acids 111-120 of human TIM3having SEQ ID NO: 286, which corresponds to amino acid residues 90-99 ofmature human TIM3 (RIQIPGIMND, SEQ ID NO: 298; see FIG. 20). In certainembodiments, an anti-TIM3 antibody binds to, or to an epitope within, aregion consisting of amino acids 58-64 of human TIM3 having SEQ ID NO:286 (CPVFECG, SEQ ID NO296) and a region consisting of amino acids111-120 of human TIM3 having SEQ ID NO: 286 (RIQIPGIMND, SEQ ID NO: 298;see FIG. 20). In certain embodiments, an anti-TIM3 antibody binds to, orto an epitope within, a region consisting of amino acids 78-89 of humanTIM3 having SEQ ID NO: 286, which corresponds to amino acid residues57-83 of mature human TIM3 (WTSRYWLNGDFR, SEQ ID NO: 297; see FIG. 20).

In one embodiment, an anti-TIM3 antibody binds to substantially the sameepitope as that of 13A3. In certain embodiments, an anti-TIM3 antibodybinds to an epitope (or region of human TIM3) comprising one or more ofamino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ IDNO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibody binds toan epitope (or region of human TIM3) comprising one or more of aminoacid residues C58, P59, F61, E62, C63, D104, R111, Q113 and D120 of SEQID NO: 286 (FIG. 20). In certain embodiments, an anti-TIM3 antibody doesnot bind significantly, or only with significantly reduced bindingaffinity, to a human TIM3 protein in which one or more of amino acidresidues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO: 286 ischanged to another amino acid, e.g., in a non-conservative amino acidsubstitution. In certain embodiments, an anti-TIM3 antibody does notbind significantly, or only with significantly reduced binding affinity,to a human TIM3 protein in which one or more of amino acid residues C58,P59, F61, E62, C63, D104, R111, Q113 and D120 of SEQ ID NO: 286 ischanged to another amino acid, e.g., in a non-conservative amino acidsubstitution.

In some embodiments, an anti-TIM3 antibody binds to substantially thesame epitope as that of 3G4. In some embodiments, an anti-TIM3 antibodybinds to an epitope (or region of human TIM3) comprising one or more ofamino acids residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQID NO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibody bindsto an epitope (or region of human TIM3) comprising one or more of aminoacid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQID NO: 286 (FIG. 20). In certain embodiments, an anti-TIM3 antibody doesnot bind significantly, or only with significantly reduced bindingaffinity, to a human TIM3 protein in which one or more of amino acidresidues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO: 286is changed to another amino acid, e.g., in a non-conservative amino acidsubstitution. In certain embodiments, an anti-TIM3 antibody does notbind significantly, or only with significantly reduced binding affinity,to a human TIM3 protein in which one or more of amino acid residues C58,P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO: 286 ischanged to another amino acid, e.g., in a non-conservative amino acidsubstitution.

In some embodiments, an anti-TIM3 antibody binds to substantially thesame epitope as that of 17C3. In certain embodiments, an anti-TIM3antibody binds to an epitope (or region of human TIM3) comprising one ormore of amino acids residues C58, P59, V60, F61, E62, C63, G64, and G116of SEQ ID NO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibodybinds to an epitope (or region of human TIM3) comprising one or more ofamino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 ofSEQ ID NO: 286 (FIG. 20). In certain embodiments, an anti-TIM3 antibodydoes not bind significantly, or only with significantly reduced bindingaffinity, to a human TIM3 protein in which one or more of amino acidresidues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO: 286is changed to another amino acid, e.g., in a non-conservative amino acidsubstitution. In certain embodiments, an anti-TIM3 antibody does notbind significantly, or only with significantly reduced binding affinity,to a human TIM3 protein in which one or more of amino acid residues C58,P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO: 286 ischanged to another amino acid, e.g., in a non-conservative amino acidsubstitution.

In some embodiments, an anti-TIM3 antibody binds to substantially thesame epitope as that of 8B9. In certain embodiments, an anti-TIM3antibody binds to an epitope (or region of human TIM3) comprising one ormore of amino acids residues L48, W78, S80, R81, W83, G86, D87, and R89of SEQ ID NO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibodybinds to an epitope (or region of human TIM3) comprising one or more ofamino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 ofSEQ ID NO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibodybinds to substantially the same epitope as that of 8B9. In certainembodiments, an anti-TIM3 antibody binds to an epitope (or region ofhuman TIM3) comprising one or more of amino acids residues L48, W78,S80, R81, W83, G86, D87, R89, and D104 of SEQ ID NO: 286 (FIG. 20). Incertain embodiments, an anti-TIM3 antibody does not bind significantly,or only with significantly reduced binding affinity, to a human TIM3protein in which one or more of amino acid residues L48, W78, S80, R81,W83, G86, D87, and R89 of SEQ ID NO: 286 is changed to another aminoacid, e.g., in a non-conservative amino acid substitution. In certainembodiments, an anti-TIM3 antibody does not bind significantly, or onlywith significantly reduced binding affinity, to a human TIM3 protein inwhich one or more of amino acid residues L48, W78, S80, R81, W83, L84,G86, D87, and R89 of SEQ ID NO: 286 is changed to another amino acid,e.g., in a non-conservative amino acid substitution. In certainembodiments, an anti-TIM3 antibody does not bind significantly, or onlywith significantly reduced binding affinity, to a human TIM3 protein inwhich one or more of amino acid residues L48, W78, S80, R81, W83, G86,D87, R89, and D104 of SEQ ID NO: 286 (FIG. 20) is changed to anotheramino acid, e.g., in a non-conservative amino acid substitution.

In certain embodiments, anti-TIM3 antibodies compete for binding tohuman TIM3 with (or inhibit binding of) anti-TIM3 antibodies comprisingCDRs or variable regions described herein, e.g., those of antibodies13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 and any of TIM3.2 to TIM3.18. Incertain embodiments, anti-TIM3 antibodies inhibit binding of antibodies13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any of TIM3.2 to TIM3.18 tohuman TIM3 by at least 50%, 60%, 70%, 80%, 90% or by 100%. In certainembodiments, 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any of TIM3.2 toTIM3.18 inhibit binding of anti-TIM3 antibodies to human TIM3 by atleast 50%, 60%, 70%, 80%, 90% or by 100%. In certain embodiments,anti-TIM3 antibodies inhibit binding of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9,8C4 or any of TIM3.2 to TIM3.18 to human TIM3 by at least 50%, 60%, 70%,80%, 90% or by 100% and 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any ofTIM3.2 to TIM3.18 inhibit binding of the anti-TIM3 antibodies to humanTIM3 by at least 50%, 60%, 70%, 80%, 90% or by 100% (e.g., compete inboth directions).

VII. Engineered and Modified Antibodies

Also provided are engineered and modified antibodies that can beprepared using an antibody having one or more of the VH and/or VLsequences disclosed herein as starting material to engineer a modifiedantibody, which modified antibody can have altered properties from thestarting antibody. An antibody can be engineered by modifying one ormore residues within one or both variable regions (i.e., VH and/or VL),for example within one or more CDR regions and/or within one or moreframework regions. Additionally or alternatively, an antibody can beengineered by modifying residues within the constant region(s), forexample to alter the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.).

Accordingly, another embodiment described herein pertains to an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencescomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 41-45, SEQ ID NOs: 46-52, 122-125, and SEQ ID NOs: 53-59,126-129, respectively, and a light chain variable region comprisingCDR1, CDR2, and CDR3 sequences comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 64-65, SEQ ID NOs:66-67, and SEQ ID NOs: 68-71, respectively. Thus, such antibodiescontain the VH and VL CDR sequences of monoclonal antibodies 13A3, 3G4,17C3, 17C8, 9F6, 8B9, 8C4 or any one of TIM3.2 to TIM3.18, yet cancontain different framework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops”/.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

In some embodiments, the framework sequences for use in the anti-TIM3antibodies described herein are those that are structurally similar tothe framework sequences used by the anti-TIM3 antibodies describedherein. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3sequences, can be grafted onto framework regions that have the identicalsequence as that found in the germline immunoglobulin gene from whichthe framework sequence derive, or the CDR sequences can be grafted ontoframework regions that contain one or more mutations as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al).

Engineered anti-TIM3 antibodies described herein include those in whichmodifications have been made to framework residues within VH and/or VL,e.g., to improve the properties of the antibody, e.g., a mutation atamino acid 107 in 9F6. Typically such framework modifications are madeto decrease the immunogenicity of the antibody. For example, oneapproach is to “backmutate” one or more framework residues to thecorresponding germline sequence. More specifically, an antibody that hasundergone somatic mutation can contain framework residues that differfrom the germline sequence from which the antibody is derived. Suchresidues can be identified by comparing the antibody framework sequencesto the germline sequences from which the antibody is derived. To returnthe framework region sequences to their germline configuration, thesomatic mutations can be “backmutated” to the germline sequence by, forexample, site-directed mutagenesis or PCR-mediated mutagenesis. Such“backmutated” antibodies are also intended to be encompassed. Anothertype of framework modification involves mutating one or more residueswithin the framework region, or even within one or more CDR regions, toremove T cell epitopes to thereby reduce the potential immunogenicity ofthe antibody. This approach is also referred to as “deimmunization” andis described in further detail in U.S. Patent Publication No.20030153043 by Carr et al.

Another type of variable region modification is to mutate amino acidresidues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. In some embodiments, conservative modifications (asdiscussed above) are introduced. The mutations can be amino acidsubstitutions, additions or deletions. Moreover, typically no more thanone, two, three, four or five residues within a CDR region are altered.

Accordingly, also provided are isolated anti-TIM3 monoclonal antibodies,or antigen binding portions thereof, comprising a heavy chain variableregion comprising:

(a) a VH CDR1 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 41-45, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 41-45;(b) a VH CDR2 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 46-52 and 122-125, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 46-52 and 122-125;(c) a VH CDR3 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 53-59 and 126-129, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 53-59 and 126-129;(d) a VL CDR1 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 64-65, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 64-65;(e) a VL CDR2 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 66-67, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 66-67; and(f) a VL CDR3 region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 68-71, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 68-71.

Methionine residues in CDRs of antibodies can be oxidized, resulting inpotential chemical degradation and consequent reduction in potency ofthe antibody. Accordingly, also provided are anti-TIM3 antibodies whichhave one or more methionine residues in the heavy and/or light chainCDRs replaced with amino acid residues which do not undergo oxidativedegradation. In one embodiment, the methionine residues in the CDRs ofantibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any of TIM3.2 toTIM3.18 are replaced with amino acid residues which do not undergooxidative degradation.

Similarly, deamidation sites can be removed from anti-TIM3 antibodies,particularly in the CDRs.

Anti-TIM3 variable regions described herein can be linked (e.g.,covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3 or IgG4Fc, which can be of any allotype or isoallotype, e.g., for IgG1: G1m,G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3:G3m, G3m21(g1), G3m28(g5), G3 m1 1(b0), G3m5(b1), G3m13(b3), G3m14(b4),G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v);and for K: Km, Km1, Km2, Km3 (see, e.g., Jefferies et al. (2009) mAbs1:1).

In certain embodiments, anti-TIM3 variable regions described herein arelinked to an effectorless or mostly effectorless Fc, e.g., IgG1.

Generally, variable regions described herein can be linked to an Fccomprising one or more modification, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody described herein can bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, to alter oneor more functional properties of the antibody. Each of these embodimentsis described in further detail below. The numbering of residues in theFc region is that of the EU index of Kabat.

The Fc region encompasses domains derived from the constant region of animmunoglobulin, including a fragment, analog, variant, mutant orderivative of the constant region. Suitable immunoglobulins includeIgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE and IgM,The constant region of an immunoglobulin is defined as anaturally-occurring or synthetically-produced polypeptide homologous tothe immunoglobulin C-terminal region, and can include a CH1 domain, ahinge, a CH2 domain, a CH3 domain, or a CH4 domain, separately or incombination.

Ig molecules interact with multiple classes of cellular receptors. Forexample IgG molecules interact with three classes of Fcγ receptors(FcγR) specific for the IgG class of antibody, namely FcγRI, FcγRII, andFcγRIII. The important sequences for the binding of IgG to the FcγRreceptors have been reported to be located in the CH2 and CH3 domains.The serum half-life of an antibody is influenced by the ability of thatantibody to bind to an Fc receptor (FcR).

In certain embodiments, the Fc region is a variant Fc region, e.g., anFc sequence that has been modified (e.g., by amino acid substitution,deletion and/or insertion) relative to a parent Fc sequence (e.g., anunmodified Fc polypeptide that is subsequently modified to generate avariant), to provide desirable structural features and/or biologicalactivity,

Generally, variants of the constant region or portions thereof, e.g.,CH1, CL, hinge, CH2 or CH3 domains can comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more mutations, and/or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1mutation, or 1-10 or 1-5 mutations, or comprise an amino acid sequencethat is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to that of the corresponding wild-type region or domain (CH1,CL, hinge, CH2, or CH3 domain, respectively), provided that the heavychain constant region comprising the specific variant retains thenecessary biological activity.

For example, one can make modifications in the Fc region in order togenerate an Fc variant that (a) has increased or decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased ordecreased complement mediated cytotoxicity (CDC), (c) has increased ordecreased affinity for C1q and/or (d) has increased or decreasedaffinity for a Fc receptor relative to the parent Fc. Such Fc regionvariants will generally comprise at least one amino acid modification inthe Fc region. Combining amino acid modifications is thought to beparticularly desirable. For example, the variant Fc region can includetwo, three, four, five, etc. substitutions therein, e.g., of thespecific Fc region positions identified herein.

A variant Fc region can also comprise a sequence alteration whereinamino acids involved in disulfide bond formation are removed or replacedwith other amino acids. Such removal can avoid reaction with othercysteine-containing proteins present in the host cell used to producethe anti-TIM3 antibodies described herein. Even when cysteine residuesare removed, single chain Fc domains can still form a dimeric Fc domainthat is held together non-covalently. In other embodiments, the Fcregion can be modified to make it more compatible with a selected hostcell. For example, one can remove the PA sequence near the N-terminus ofa typical native Fc region, which can be recognized by a digestiveenzyme in E. coli such as proline iminopeptidase. In other embodiments,one or more glycosylation sites within the Fc domain can be removed.Residues that are typically glycosylated (e.g., asparagine) can confercytolytic response. Such residues can be deleted or substituted withunglycosylated residues (e.g., alanine). In other embodiments, sitesinvolved in interaction with complement, such as the C1q binding site,can be removed from the Fc region. For example, one can delete orsubstitute the EKK sequence of human IgG1. In certain embodiments, sitesthat affect binding to Fc receptors can be removed, preferably sitesother than salvage receptor binding sites. In other embodiments, an Fcregion can be modified to remove an ADCC site. ADCC sites are known inthe art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) withregard to ADCC sites in IgG1. Specific examples of variant Fc domainsare disclosed for example, in WO 97/34631 and WO 96/32478.

In one embodiment, the hinge region of Fc is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of Fc is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody. In one embodiment, the Fc hinge region of an antibody ismutated to decrease the biological half-life of the antibody. Morespecifically, one or more amino acid mutations are introduced into theCH2-CH3 domain interface region of the Fc-hinge fragment such that theantibody has impaired Staphylococcyl protein A (SpA) binding relative tonative Fc-hinge domain SpA binding. This approach is described infurther detail in U.S. Pat. No. 6,165,745 by Ward et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320, 322, 330, and/or 331 can be replaced with a different amino acidresidue such that the antibody has an altered affinity for an effectorligand but retains the antigen-binding ability of the parent antibody.The effector ligand to which affinity is altered can be, for example, anFc receptor or the C1 component of complement. This approach isdescribed in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260,both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region can be modified to decreaseantibody dependent cellular cytotoxicity (ADCC) and/or to decrease theaffinity for an Fcγ receptor by modifying one or more amino acids at thefollowing positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245,247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322,324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340,360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434,435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D,239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variantsinclude 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F,267E/324T, and 267E/268F7324T. Other modifications for enhancing FcγRand complement interactions include but are not limited to substitutions298 A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L,292P, 300L, 396L, 3051, and 396L. These and other modifications arereviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

Fc modifications that increase binding to an Fcγ receptor include aminoacid modifications at any one or more of amino acid positions 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279,280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303,305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 338, 340, 360, 373,376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439 of the Fc region, wherein the numbering of the residues in the Fcregion is that of the EU index as in abat (WO00/42072).

Other Fc modifications that can be made to Fcs are those for reducing orablating binding to FcγR and/or complement proteins, thereby reducing orablating Fc-mediated effector functions such as ADCC, ADCP, and CDC.Exemplary modifications include but are not limited substitutions,insertions, and deletions at positions 234, 235, 236, 237, 267, 269,325, 328, 330, and/or 331 (e.g., 330 and 331), wherein numbering isaccording to the EU index. Exemplary substitutions include but are notlimited to 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S, and331S (e.g., 330S, and 331S), wherein numbering is according to the EUindex. An Fc variant can comprise 236R/328R. Other modifications forreducing FcγR and complement interactions include substitutions 297A,234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331 S, 220S, 226S,229S, 238S, 233P, and 234V, as well as removal of the glycosylation atposition 297 by mutational or enzymatic means or by production inorganisms such as bacteria that do not glycosylate proteins. These andother modifications are reviewed in Strohl, 2009, Current Opinion inBiotechnology 20:685-691.

Optionally, the Fc region can comprise a non-naturally occurring aminoacid residue at additional and/or alternative positions known to oneskilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375;6,737,056; 6,194,551; 7,317,091; 8,101,720; PCX Patent Publications WO00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO05/040217, WO 05/092925 and WO 06/020114).

Fc variants that enhance affinity for an inhibitory receptor FcγRIIb canalso be used. Such variants can provide an Fc fusion protein withimmunomodulatory activities related to FcγRIIb cells, including forexample B cells and monocytes. In one embodiment, the Fc variantsprovide selectively enhanced affinity to FcγRIIb relative to one or moreactivating receptors. Modifications for altering binding to FcγRIIbinclude one or more modifications at a position selected from the groupconsisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327,328, 330, 331, and 332, according to the EU index. Exemplarysubstitutions for enhancing FcγRllb affinity include but are not limitedto 234A, 234D, 234E, 234F, 234W, 235D, 235E, 235F, 235R, 235Y, 236D,236N, 237A, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D,327E, 328F, 328W, 328Y, 330S, 331S, and 332E. Exemplary substitutionsinclude 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y.Other Fc variants for enhancing binding to FcγRIIb include 235Y/267E,236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F.

The affinities and binding properties of an Fc region for its ligand canbe determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art including but not limitedto, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay(ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACOREanalysis), and other methods such as indirect binding assays,competitive inhibition assays, fluorescence resonance energy transfer(FRET), gel electrophoresis and chromatography (e.g., gel filtration).These and other methods can utilize a label on one or more of thecomponents being examined and/or employ a variety of detection methodsincluding but not limited to chromogenic, fluorescent, luminescent, orisotopic labels. A detailed description of binding affinities andkinetics can be found in Paul, W. E., ed., Fundamental immunology, 4thEd., Lippincott-Raven, Philadelphia (1999), which focuses onantibody-immunogen interactions.

In certain embodiments, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, thiscan be done by increasing the binding affinity of the Fc region forFcRn, For example, one or more of more of following residues can bemutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No.6,277,375. Specific exemplary substitutions include one or more of thefollowing: T252L, T254S, and/or T256F. Alternatively, to increase thebiological half life, the antibody can be altered within the CH1 or CLregion to contain a salvage receptor binding epitope taken from twoloops of a CH2 domain of an Fc region of an IgG, as described in U.S.Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplaryvariants that increase binding to FcRn and/or improve pharmacokineticproperties include substitutions at positions 259, 308, 428, and 434,including for example 2591, 308F, 428L, 428M, 434S, 4341 1. 434F, 434Y,and 434X1. Other variants that increase Fc binding to FcRn include:250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al. 2004, J. Biol. Chem.279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A,378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry,2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q,256E, 256D, 256T, 309P, 311S, 433R, 433S, 433I, 433P, 433Q, 434H, 434F,434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311S (Dall Acqua et al.Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006,Journal of Biological Chemistry 281:23514-23524). Other modificationsfor modulating FcRn binding are described in Yeung et al., 2010, JImmunol, 182:7663-7671.

In certain embodiments, hybrid IgG isotypes with particular biologicalcharacteristics can be used. For example, an IgG1/IgG3 hybrid variantcan be constructed by substituting IgG1 positions in the CH2 and/or CH3region with the amino acids from IgG3 at positions where the twoisotypes differ. Thus a hybrid variant IgG antibody can be constructedthat comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T,356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In other embodimentsdescribed herein, an IgG1/IgG2 hybrid variant can be constructed bysubstituting IgG2 positions in the CH2 and/or CH3 region with aminoacids from IgG1 at positions where the two isotypes differ. Thus ahybrid variant IgG antibody can be constructed that comprises one ormore substitutions, e.g., one or more of the following amino acidsubstitutions: 233E, 234L, 235L, −236G (referring to an insertion of aglycine at position 236), and 327A.

Moreover, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A, which hasbeen shown to exhibit enhanced FcγRIIIa binding and ADCC activity(Shields et al., 2001). Other IgG1 variants with strongly enhancedbinding to FcγRIIIa have been identified, including variants withS239D/I332E and S239D/I332E/A330L mutations which showed the greatestincrease in affinity for FcγRIIIa, a decrease in FcγRIIb binding, andstrong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006).Introduction of the triple mutations into antibodies such as alemtuzumab(CD52-specific), trastuzumab (HER2/neu-specific), rituximab(CD20-specific), and cetuximab (EGFR-specific) translated into greatlyenhanced ADCC activity in vitro, and the S239D/I332E variant showed anenhanced capacity to deplete B cells in monkeys (Lazar et al., 2006). Inaddition, IgG1 mutants containing L235V, F243L, R292P, Y300L and P396Lmutations which exhibited enhanced binding to FcγRIIIa and concomitantlyenhanced ADCC activity in transgenic mice expressing human FcγRIIIa inmodels of B cell malignancies and breast cancer have been identified(Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutantsthat can be used include: S298A/E333A/L334A, S239D/I332E,S239D/I332E/A330L, L235V/F243L/R292P/Y300L/P396L, and M428L/N434S.

In certain embodiments, an Fc is chosen that has reduced binding toFcγRs. An exemplary Fc, e.g., IgG1 Fc, with reduced FcγR bindingcomprises the following three amino acid substitutions: L234A, L235E andG237A.

In certain embodiments, an Fc is chosen that has reduced complementfixation. An exemplary Fc, e.g., IgG1 Fc, with reduced complementfixation has the following two amino acid substitutions: A330S andP331S.

In certain embodiments, an Fc is chosen that has essentially no effectorfunction, i.e., it has reduced binding to FcγRs and reduced complementfixation. An exemplary Fc, e.g., IgG1 Fc, that is effectorless comprisesthe following five mutations: L234A, L235E, G237A, A330S and P331S.

When using an IgG4 constant domain, it can include the substitutionS228P, which mimics the hinge sequence in IgG1 and thereby stabilizesIgG4 molecules.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation canincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Glycosylation of the constant region on N297 can be prevented bymutating the N297 residue to another residue, e.g., N297A, and/or bymutating an adjacent amino acid, e.g., 298 to thereby reduceglycosylation on N297.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant anti-TIM3 antibodies described herein to thereby produce anantibody with altered glycosylation. For example, EP 1,176,195 by Hanaiet al. describes a cell line with a functionally disrupted FUT8 gene,which encodes a fucosyl transferase, such that antibodies expressed insuch a cell line exhibit hypofucosylation. PCT Publication WO 03/035835by Presta describes a variant CHO cell line, Led 3 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases{e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).

Another modification of the anti-TIM3 antibodies described herein ispegylation. An antibody can be pegylated to, for example, increase thebiological (e.g., serum) half-life of the antibody. To pegylate anantibody, the antibody, or fragment thereof, typically is reacted withpolyethylene glycol (PEG), such as a reactive ester or aldehydederivative of PEG, under conditions in which one or more PEG groupsbecome attached to the antibody or antibody fragment. In someembodiments, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (CI-CIO) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the anti-TIM3 antibodies described herein. See for example, EP 0 154316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

In some embodiments, an anti-TIM3 antibody comprises a heavy chainconstant region and a light chain constant region, wherein the heavychain constant region is selected from the group consisting of SEQ IDNOs: 263-266.

VIII. Antibody Physical Properties

Anti-TIM3 antibodies, e.g., those described herein, have some or all ofthe physical characteristics of the specific anti-TIM3 antibodiesdescribed herein, such as the characteristics described in the Examples.

Anti-TIM3 antibodies described herein can contain one or moreglycosylation sites in either the light or heavy chain variable region.Such glycosylation sites can result in increased immunogenicity of theantibody or an alteration of the pK of the antibody due to alteredantigen binding (Marshall et al., (1972) Annu Rev Biochem 41:673-702;Gala and Morrison (2004) J. Immunol 172:5489-94; Wallick et al., (1988)J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh etal., (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol37:697-706). Glycosylation has been known to occur at motifs containingan N-X-S/T sequence. In some instances, an anti-TIM3 antibody does notcontain variable region glycosylation. This can be achieved either byselecting antibodies that do not contain the glycosylation motif in thevariable region or by mutating residues within the glycosylation region.

In certain embodiments, the anti-TIM3 antibodies described herein do notcontain asparagine isomerism sites. The deamidation of asparagine canoccur on N-G or D-G sequences and result in the creation of anisoaspartic acid residue that introduces a kink into the polypeptidechain and decreases its stability (isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pi), which generallyfalls in the pH range between 6 and 9.5. The pi for an IgG1 antibodytypically falls within the pH range of 7-9.5 and the pi for an IgG4antibody typically falls within the pH range of 6-8. There isspeculation that antibodies with a pi outside the normal range can havesome unfolding and instability under in vivo conditions. Thus, ananti-TIM3 antibody can contain a pi value that falls in the normalrange. This can be achieved either by selecting antibodies with a pi inthe normal range or by mutating charged surface residues.

Each antibody will have a characteristic melting temperature, with ahigher melting temperature indicating greater overall stability in vivo(Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71).Generally, the T_(M)i (the temperature of initial unfolding) can begreater than 60° C., greater than 65° C., or greater than 70° C. Themelting point of an antibody can be measured using differential scanningcalorimetry (Chen et al., (2003) Pharm Res 20: 1952-60; Ghirlando etal., (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al.,(2002) J. Chromatogr Sci 40:343-9).

In one embodiment, antibodies are selected that do not degrade rapidly.Degradation of an antibody can be measured using capillaryelectrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995)Anal Chem 67:3626-32).

In another embodiment, antibodies are selected that have minimalaggregation effects, which can lead to the triggering of an unwantedimmune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, 20% or less, 15% or less, 10% or less, or 5% or less.Aggregation can be measured by several techniques, includingsize-exclusion column (SEC), high performance liquid chromatography(HPLC), and light scattering.

In certain embodiments, an anti-TIM3 antibody has a combination ofstructures and properties described in sections (I), (II), (III), (IV),(V), (VI), (VII), (VIII), and (IX) above. In one embodiment, ananti-TIM3 antibody cross-competes with Antibodies 13A3, 17C3, 8B9, 8C4,3G4, 17C8, and 9F6, as described in Sections I and/or VI, derived fromthe germline sequence as described in Section III, has conservedmutations as described in Section V, and/or has homology to theanti-TIM3 antibodies in Section I and II as described in Section IV incombination with one or more functional properties described anywhereherein.

IX. Methods of Engineering Antibodies

As discussed above, the anti-TIM3 antibodies having VH and VL sequencesdisclosed herein can be used to create new anti-TIM3 antibodies bymodifying the VH and/or VL sequences, or the constant region(s) attachedthereto. Thus, in another aspect described herein, the structuralfeatures of an anti-TIM3 antibody described herein are used to createstructurally related anti-TIM3 antibodies that retain at least onefunctional property of the anti-TIM3 antibodies described herein, suchas binding to human TIM3 and cynomolgus TIM3. For example, one or moreCDR regions of 17C3, 8B9, 8C4, 3G4, 17C8, 9F6, 13A3, or any of TIM3.2 toTIM3.18 can be combined recombinantly with known framework regionsand/or other CDRs to create additional, recombinantly-engineered,anti-TIM3 antibodies described herein, as discussed above. Other typesof modifications include those described in the previous section. Thestarting material for the engineering method is one or more of the VHand/or VL sequences provided herein, or one or more CDR regions thereof.To create the engineered antibody, it is not necessary to actuallyprepare (i.e., express as a protein) an antibody having one or more ofthe VH and/or VL sequences provided herein, or one or more CDR regionsthereof. Rather, the information contained in the sequence(s) is used asthe starting material to create a “second generation” sequence(s)derived from the original sequence(s) and then the “second generation”sequence(s) is prepared and expressed as a protein.

Accordingly, provided herein are methods for preparing an anti-TIM3antibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 sequence selected from the group consisting of SEQ IDNOs: 41 to 45, a CDR2 sequence selected from the group consisting of SEQID NOs: 46 to 52 and 122-125, and/or a CDR3 sequence selected from thegroup consisting of SEQ ID NOs: 53 to 59 and 126-129; and (ii) a lightchain variable region antibody sequence comprising a CDR1 sequenceselected from the group consisting of SEQ ID NOs: 64 and 65, a CDR2sequence selected from the group consisting of SEQ ID NOs: 66 and 67,and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs:68 to 71;(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and(c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. In some embodiments, the antibody encodedby the altered antibody sequence(s) is one that retains one, some or allof the functional properties of the anti-TIM3 antibodies describedherein, which include:

(1) binding to soluble human TIM3, e.g., with a KD of 10 nM or less(e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;

(2) binding to soluble cynomolgus TIM3, e.g., with a KD of 100 nM orless (e.g., 0.01 nM to 100 nM), e.g., as measured by Biacore, e.g., asdescribed in the Examples;

(3) binding to membrane bound human TIM3, e.g., with an EC50 of 1 ug/mLor less (e.g., 0.01 ug/mL to 1 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);

(4) binding to membrane bound human TIM3, e.g., with a K_(D) of 1 nM orless (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard analysis,e.g., as described in the Examples;

(5) binding to membrane bound cynomolgus TIM3, e.g., with an EC50 of 20ug/mL or less (e.g., 0.01 ug/mL to 20 ug/mL), e.g., as measured by flowcytometry (e.g., as described in the Examples);

(6) binding to membrane bound cynomolgus TIM3, e.g., with a K_(D) of 1nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchardanalysis, e.g., as described in the Examples;

(7) inducing or enhancing T cell activation (e.g., by blocking orreducing the inhibitory effect of TIM3), as evidenced by (i) increasedIFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs)and/or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1cells or TILs), e.g., as described in the Examples;(8) stimulating T cell proliferation in a mixed lymphocyte reaction(MLR) assay, e.g., as described in the Examples;(9) inhibiting the binding of phosphatidylserine to TIM3, e.g., asmeasured by PS-hTIM3 “in-tandem” blocking assay, e.g., as described inthe Examples;(10) not internalizing or downregulating cell surface TIM3 when bindingto TIM3 on cells;(11) binding to one of the following regions of human TIM3 extracellulardomain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND(SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298,respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297), e.g., as describedin the Examples;(12) having reduced binding to human TIM3 in which one or more of aminoacids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84,G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (as numbered inSEQ ID NO: 286 (FIG. 20)) is substituted with another amino acidrelative to binding to wildtype human TIM3, e.g., as described in theExamples;(13) competing in either direction or both directions for binding tohuman TIM3 with an antibody comprising VH and VL domains of any one of13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or TIM3.7, TIM3.8, TIM3.10,TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, andTIM3.18, e.g., as described in the Examples;(14) binding to human TIM3 regions ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367)and ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) as determined by HDX-MS,e.g., as described in the Examples;(15) having the heavy chain and/or light chain variable regions interactwith at least 5, 10, 15, 20 or all of the following amino acids of humanTIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68,R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionallyT70 and/or I112, as determined by X-ray crystallography (e.g., describedin the Examples; numbering per SEQ ID NO: 286 (FIG. 20)); and/or(16) (a) having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6,7, 8 or 9 of amino acids C58, P59, F61, E62, C63, R111, D120, andoptionally D104 and Q113 (numbering per SEQ ID NO: 286 (FIG. 20)) aresubstituted with another amino acid relative to binding to wildtypehuman TIM3; (b) binding to ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367),¹¹¹RIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 368) and ¹¹⁹NDEKFNLKL¹²⁷ (SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and/or (c)competing with or cross-blocking with the binding of 13A3 orTIM3.18.IgG1.3, e.g., as described in the Examples.

The altered antibody can exhibit one or more, two or more, three ormore, four or more, five or more, six or more, seven or more, eight ormore, nine or more, or all of the functional properties set forth as (1)through (16) above. The functional properties of the altered antibodiescan be assessed using standard assays available in the art and/ordescribed herein, such as those set forth in the Examples (e.g., ELISAs,FACS).

In certain embodiments of the methods of engineering the anti-TIM3antibodies described herein, mutations can be introduced randomly orselectively along all or part of an anti-TIM3 antibody coding sequenceand the resulting modified anti-TIM3 antibodies can be screened forbinding activity and/or other functional properties as described herein.Mutational methods have been described in the art. For example, PCTPublication WO 02/092780 by Short describes methods for creating andscreening antibody mutations using saturation mutagenesis, syntheticligation assembly, or a combination thereof. Alternatively, PCTPublication WO 03/074679 by Lazar et al. describes methods of usingcomputational screening methods to optimize physiochemical properties ofantibodies.

X. Nucleic Acid Molecules

Another aspect described herein pertains to nucleic acid molecules thatencode the anti-TIM3 antibodies described herein. The nucleic acids canbe present in whole cells, in a cell lysate, or in a partially purifiedor substantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids (e.g., otherchromosomal DNA, e.g., the chromosomal DNA that is linked to theisolated DNA in nature) or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, restrictionenzymes, agarose gel electrophoresis and others well known in the art.See, F. Ausubel, et al., ed. (1987) Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York. A nucleicacid described herein can be, for example, DNA or RNA and can or cannotcontain intronic sequences. In a certain embodiments, the nucleic acidis a cDNA molecule.

Nucleic acids described herein can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromthe library.

Some nucleic acids molecules described herein are those encoding the VHand VL sequences of the 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8 or any ofTIM3.2 to TIM3.18 antibodies. Exemplary DNA sequences encoding the VHsequences of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, and 17C8 are set forth inSEQ ID NOs: 167 to 173, 245 to 254, and 359. Exemplary DNA sequencesencoding the VL sequences of 13A3, 17C3, and 3G4 are set forth in SEQ IDNO: 193. Exemplary DNA sequences encoding the VL sequences of 8B9, 8C4,and 17C8 are set forth in SEQ ID NO: 194. Exemplary DNA sequencesencoding the VL sequences of 9F6 are set forth in SEQ ID NOs: 194 to196. Exemplary DNA sequences encoding the heavy chain sequences of 13A3,8B9, 8C4, 17C3, 9F6, 3G4, and 17C8 are set forth in SEQ ID NOs: 134 to161, 205 to 244, and 355-358. Exemplary DNA sequences encoding the lightchain sequences of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, and 17C8 are setforth in SEQ ID NOs: 162-166.

Exemplary nucleic acids encoding the mature VH and VL domains of13A3.IgG1.1 and 13A3.IgG1.3 (same variable region) antibodies are setforth as SEQ ID NOs: 167 and 193, respectively. Exemplary nucleic acidsencoding the mature heavy chains of 13A3.IgG1.1 and 13A3.IgG1.3antibodies are set forth as SEQ ID NOs: 134 and 148, respectively, andan exemplary nucleic acid encoding the mature light chain of 13A3.IgG1.1and 13A3.IgG1.3 antibodies is set forth as SEQ ID NO: 162.

Exemplary nucleic acids encoding the mature VH and VL domains of8B9.IgG1.1 and 8B9.IgG1.3 (same variable region) antibodies are setforth as SEQ ID NOs: 168 and 194, respectively. Exemplary nucleic acidsencoding the mature heavy chains of 8B9.IgG1.1 and 8B9.IgG1.3 antibodiesare set forth as SEQ ID NOs: 135 and 149, respectively, and an exemplarynucleic acid encoding the mature light chain of 8B9.IgG1.1 and8B9.IgG1.3 antibodies is set forth as SEQ ID NO: 163.

Exemplary nucleic acids encoding the mature VH and VL domains of8C4.IgG1.1 and 8C4.IgG1.3 (same variable region) antibodies are setforth as SEQ ID NOs: 169 and 194, respectively. Exemplary nucleic acidsencoding the mature heavy chains of 8C4.IgG1.1 and 8C4.IgG1.3 antibodiesare set forth as SEQ ID NOs: 136 and 150, respectively, and an exemplarynucleic acid encoding the mature light chain of 8C4.IgG1.1 and8C4.IgG1.3 antibodies is set forth as SEQ ID NO: 163.

Exemplary nucleic acids encoding the mature VH and VL domains of17C3.IgG1.1 and 17C3.IgG1.3 (same variable region) antibodies are setforth as SEQ ID NOs: 170 and 193, respectively. Exemplary nucleic acidsencoding the mature heavy chains of 17C3.IgG1.1 and 17C3.IgG1.3antibodies are set forth as SEQ ID NOs: 137 and 151, respectively, andan exemplary nucleic acid encoding the mature light chain of 17C3.IgG1.1and 17C3.IgG1.3 antibodies is set forth as SEQ ID NO: 162.

Exemplary nucleic acids encoding the mature VH and VL domains of9F6.IgG1.1 and 9F6.IgG1.3 (same variable region) antibodies are setforth as SEQ ID NOs: 171 and 197, respectively. Exemplary nucleic acidsencoding the mature heavy chains of 9F6.IgG1.1 and 9F6.IgG1.3 antibodiesare set forth as SEQ ID NOs: 138 and 152, respectively, and an exemplarynucleic acid encoding the mature light chain of 9F6.IgG1.1 and9F6.IgG1.3 antibodies is set forth as SEQ ID NO: 166.

Exemplary nucleic acids encoding the mature VH and VL domains of3G4.IgG1.1 and 3G4.IgG1.3 (same variable region) antibodies are setforth as SEQ ID NOs: 172 and 193, respectively. Exemplary nucleic acidsencoding the mature heavy chains of 3G4.IgG1.1 and 3G4.IgG1.3 antibodiesare set forth as SEQ ID NOs: 139 and 153, respectively, and an exemplarynucleic acid encoding the mature light chain of 3G4.IgG1.1 and3G4.IgG1.3 antibodies is set forth as SEQ ID NO: 162.

Exemplary nucleic acids encoding the mature VH and VL domains of17C8.IgG1.1 and 17C8.IgG1.3 (same variable region) antibodies are setforth as SEQ ID NOs: 173 and 194, respectively. Exemplary nucleic acidsencoding the mature heavy chains of 17C8.IgG1.1 and 17C8.IgG1.3antibodies are set forth as SEQ ID NOs: 140 and 154, respectively, andan exemplary nucleic acid encoding the mature light chain of 17C8.IgG1.1and 17C8.IgG1.3 antibodies is set forth as SEQ ID NO: 163.

The above exemplary nucleic acids can further include a signal peptideset forth in SEQ ID NOs: 267 to 271 and 361. The nucleotide sequencesencoding these signal peptides are set forth as SEQ ID NOs: 272 to 276,362, and 363.

The nucleic acid molecules described herein may be modified to deletespecific sequences, e.g., restriction enzyme recognition sequences, orto optimize codons.

A method for making 13A3 IgG1.1, 8B9 IgG1.1, 8C4 IgG1.1, 17C3 IgG1.1,9F6 IgG1.1, 3G4 IgG1.1, 17C8 IgG1.1 and/or TIM3.2 to TIM3.18 IgG1.1 cancomprise expressing the heavy chain and the light chains in a cell linecomprising the nucleotide sequences encoding the heavy and light chainswith a signal peptide, e.g., for 13A3 IgG1.1, SEQ ID NOs: 269 and 268,respectively. A method for making 13A3 IgG1.3, 8B9 IgG1.3, 8C4 IgG1.3,17C3 IgG1.3, 9F6 IgG1.3, 3G4 IgG1.3, and/or 17C8 IgG1.3 can compriseexpressing the heavy chain and the light chains in a cell linecomprising the nucleotide sequences encoding the heavy and light chainswith a signal peptide, e.g., for 13A3 IgG1.3, SEQ ID NOs: 274 and 273,respectively. Host cells comprising these nucleotide sequences areencompassed herein.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (hinge,CH1, CH2, and/or CH3). The sequences of human heavy chain constantregion genes are known in the art (see e.g., Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standardPCR amplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, for example, an IgG1region. For a Fab fragment heavy chain gene, the VH-encoding DNA can beoperatively linked to another DNA molecule encoding only the heavy chainCH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Also provided herein are nucleic acid molecules encoding VH and VLsequences that are homologous to those of the 17C3, 8B9, 8C4, 3G4, 17C8,9F6, 13A3 and any of TIM3.2 to TIM3.18 antibodies. Exemplary nucleicacid molecules encode VH and VL sequences that are at least 70%identical, for example, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identical, to nucleic acidmolecules encoding the VH and VL sequences of the 17C3, 8B9, 8C4, 3G4,17C8, 9F6, 13A3 or any of TIM3.2 to TIM3.18 antibodies. Also providedherein are nucleic acid molecules with conservative substitutions (i.e.,substitutions that do not alter the resulting amino acid sequence upontranslation of nucleic acid molecule), e.g., for codon optimization.

Also provided are nucleic acids encoding the VH and/or VL regions ofanti-TIM3 antibodies, such as the anti-TIM3 antibodies described herein,which nucleic acids comprise a nucleotide sequence that is at leastabout 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any ofthe nucleotide sequences encoding the VH and/or VL regions of anti-TIM3antibodies described herein.

Also provided are nucleic acids encoding the heavy chain and/or thelight chain of anti-TIM3 antibodies, such as the anti-TIM3 antibodiesdescribed herein, which nucleic acids comprise a nucleotide sequencethat is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to any of the nucleotide sequences encoding the heavy and/orlight chains of anti-TIM3 antibodies described herein.

XI. Antibody Production

Monoclonal anti-TIM3 antibodies described herein can be produced using avariety of known techniques, such as the standard somatic cellhybridization technique described by Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures arepreferred, in principle, other techniques for producing monoclonalantibodies also can be employed, e.g., viral or oncogenic transformationof B lymphocytes, phage display technique using libraries of humanantibody genes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized anti-TIM3 antibodies described herein can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al).

In one embodiment, the anti-TIM3 antibodies described herein are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstTIM3 can be generated using transgenic or transchromosomic mice carryingparts of the human immune system rather than the mouse system. Thesetransgenic and transchromosomic mice include mice referred to herein asHuMAb mice and KM mice, respectively, and are collectively referred toherein as “human Ig mice.”

The HUMAB-MOUSE® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.,(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGKmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparationand use of HuMab mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al., (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and5,770,429; all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani etal., PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO97/13852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and PCTPublication No. WO 01/14424 to Korman et al.

In certain embodiments, the anti-TIM3 antibodies described herein areraised using a mouse that carries human immunoglobulin sequences ontransgenes and transchomosomes, such as a mouse that carries a humanheavy chain transgene and a human light chain transchromosome. Suchmice, referred to herein as “KM mice”, are described in detail in PCTPublication WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-TIM3 antibodies described herein. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-TIM3 antibodies described herein. For example, mice carrying both ahuman heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-TIM3antibodies described herein.

Additional mouse systems described in the art for raising humanantibodies, e.g., human anti-TIM3 antibodies, include (i) theVELOCLMMUNE® mouse (Regeneron Pharmaceuticals, Inc.), in which theendogenous mouse heavy and light chain variable regions have beenreplaced, via homologous recombination, with human heavy and light chainvariable regions, operatively linked to the endogenous mouse constantregions, such that chimeric antibodies (human V/mouse C) are raised inthe mice, and then subsequently converted to fully human antibodiesusing standard recombinant DNA techniques; and (ii) the MEMO® mouse(Merus Biopharmaceuticals, Inc.), in which the mouse containsunrearranged human heavy chain variable regions but a single rearrangedhuman common light chain variable region. Such mice, and use thereof toraise antibodies, are described in, for example, WO 2009/15777, US2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO2011/163314, WO 2012/148873, US 2012/0070861 and US 2012/0073004.

Human monoclonal anti-TIM3 antibodies described herein can also beprepared using phage display methods for screening libraries of humanimmunoglobulin genes. Such phage display methods for isolating humanantibodies are established in the art. See for example: U.S. Pat. Nos.5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal anti-TIM3 antibodies described herein can also beprepared using SCID mice into which human immune cells have beenreconstituted such that a human antibody response can be generated uponimmunization. Such mice are described in, for example, U.S. Pat. Nos.5,476,996 and 5,698,767 to Wilson et al.

XI.A. Immunizations

To generate fully human antibodies to TIM3, transgenic ortranschromosomal mice containing human immunoglobulin genes (e.g.,HCo12, HCo7 or KM mice) can be immunized with a purified or enrichedpreparation of the TIM3 antigen and/or cells expressing TIM3 or fragmentthereof, as described for other antigens, for example, by Lonberg etal., (1994) Nature 368(6474): 856-859; Fishwild et al., (1996) NatureBiotechnology 14: 845-851 and WO 98/24884. Alternatively, mice can beimmunized with DNA encoding human TIM3 or fragment thereof. In someembodiments, the mice can be 6-16 weeks of age upon the first infusion.For example, a purified or enriched preparation (5-50 μg) of therecombinant TIM3 antigen can be used to immunize the HuMAb miceintraperitoneally. In the event that immunizations using a purified orenriched preparation of the TIM3 antigen do not result in antibodies,mice can also be immunized with cells expressing TIM3, e.g., a cellline, to promote immune responses. Exemplary cell lines includeTIM3-overexpressing stable CHO and Raji cell lines.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(IP) or subcutaneously (SC) with antigen in Ribi's adjuvant, followed byevery other week IP/SC immunizations (up to a total of 10) with antigenin Ribi's adjuvant. The immune response can be monitored over the courseof the immunization protocol with plasma samples being obtained byretroorbital bleeds. The plasma can be screened by ELISA and FACS (asdescribed below), and mice with sufficient titers of anti-TIM3 humanimmunoglobulin can be used for fusions. Mice can be boostedintravenously with antigen 3 days before sacrifice and removal of thespleen and lymph nodes. It is expected that 2-3 fusions for eachimmunization can need to be performed. Between 6 and 24 mice aretypically immunized for each antigen. Usually, HCo7, HCo12, and KMstrains are used. In addition, both HCo7 and HCo12 transgene can be bredtogether into a single mouse having two different human heavy chaintransgenes (HCo7/HCo12).

XI.B. Generation of Hybridomas Producing Monoclonal Antibodies to TIM3

To generate hybridomas producing human monoclonal anti-TIM3 antibodiesdescribed herein, splenocytes and/or lymph node cells from immunizedmice can be isolated and fused to an appropriate immortalized cell line,such as a mouse myeloma cell line. The resulting hybridomas can bescreened for the production of antigen-specific antibodies. For example,single cell suspensions of splenic lymphocytes from immunized mice canbe fused to Sp2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) withPEG. Cells can be plated in flat bottom microtiter plate, followed byincubation in selective medium. After several weeks, cells can becultured in medium. Individual wells can then be screened by ELISA forhuman monoclonal IgM and IgG antibodies. Once extensive hybridoma growthoccurs, medium can be observed usually after 10-14 days. The antibodysecreting hybridomas can be replaced, screened again, and if stillpositive for human IgG, the monoclonal antibodies can be subcloned atleast twice by limiting dilution. The stable subclones can then becultured in vitro to generate small amounts of antibody in tissueculture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored.

XI.C. Generation of Transfectomas Producing Monoclonal Antibodies toTIM3

Antibodies can be produced in a host cell transfectoma using, forexample, a combination of recombinant DNA techniques and genetransfection methods as is well known in the art (Morrison, S. (1985)Science 229: 1202).

For example, to express antibodies, or antibody fragments thereof, DNAsencoding partial or full-length light and heavy chains, can be obtainedby standard molecular biology techniques (e.g., PCR amplification orcDNA cloning using a hybridoma that expresses the antibody of interest)and the DNAs can be inserted into expression vectors such that the genesare operatively linked to transcriptional and translational controlsequences. In this context, the term “operatively linked” is intended tomean that an antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or both genes are insertedinto the same expression vector. The antibody genes are inserted intothe expression vector(s) by standard methods (e.g., ligation ofcomplementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present). Thelight and heavy chain variable regions of the anti-TIM3 antibodiesdescribed herein can be used to create full-length antibody genes of anyantibody isotype by inserting them into expression vectors alreadyencoding heavy chain constant and light chain constant regions of thedesired isotype such that the V_(H) segment is operatively linked to theC_(H) segment(s) within the vector and the V_(L) segment is operativelylinked to the C_(L) segment within the vector.

Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

In exemplary embodiments, the following signal peptides from humanantibody heavy and light chains can be used: MDWTWRVFCLLAVAPGAHS (SEQ IDNO: 267); METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268); MKHLWFFLLLVAAPRWVLS(SEQ ID NO: 269); MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 270);MDMRVPAQLLGLLLWLPGARC (SEQ ID NO: 271) or MRAWI FFLLCLAGRALA (SEQ ID NO:361). In a particular embodiment, a signal sequence used for expressionof any one of the anti-TIM3 antibodies described herein is SEQ ID NO:361. Heavy and light chains of anti-TIM3 antibodies can be expressedwith the respective signal sequence that was linked to each chain in thehybridoma from which they were cloned. Below are the signal sequences ofvarious anti-TIM3 antibodies as present in the hybridoma from which theywere cloned, which signal sequences can be used to express the sameantibody or another antibody:

(i) Amino acid sequence of 13A3 VH signal sequence: MKHLWFFLLLVAAPRWVLS(SEQ ID NO: 269)

(ii) Nucleic acid sequence of 13A3 VH signal sequence:

(SEQ ID NO: 274) ATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCC(iii) Amino acid sequence of 13A3 VL signal sequence:METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268)(iv) Nucleic acid sequence of 13A3 VL signal sequence:

(SEQ ID NO: 273) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(v) Amino acid sequence of 8B9 VH signal sequence: MKHLWFFLLLVAAPRWVLS(SEQ ID NO: 269)(vi) Nucleic acid sequence of 8B9 VH signal sequence:

(SEQ ID NO: 274) ATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCC(vii) Amino acid sequence of 8B9 VL signal sequence:METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268)(viii) Nucleic acid sequence of 8B9 VL signal sequence:

(SEQ ID NO: 273) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(ix) Amino acid sequence of 8C4 VH signal sequence: MKHLWFFLLLVAAPRWVLS(SEQ ID NO: 269)(x) Nucleic acid sequence of 8C4 VH signal sequence:

(SEQ ID NO: 274) ATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCC(xi) Amino acid sequence of 8C4 VL signal sequence: METPAQLLFLLLLWLPDTTG(SEQ ID NO: 268)(xii) Nucleic acid sequence of 8C4 VL signal sequence:

(SEQ ID NO: 273) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(xiii) Amino acid sequence of 17C3 VH signal sequence:MDWTWRVFCLLAVAPGAHS (SEQ ID NO: 267)(xiv) Nucleic acid sequence of 17C3 VH signal sequence:

(SEQ ID NO: 272) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(xv) Amino acid sequence of 17C3 VL signal sequence:METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268)(xvi) Nucleic acid sequence of 17C3 VL signal sequence:

(SEQ ID NO: 273) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(xvii) Amino acid sequence of 9F6 VH signal sequence:MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 270)(xviii) Nucleic acid sequence of 9F6 VH signal sequence:

(SEQ ID NO: 275) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(xix) Amino acid sequence of 9F6 VL1 signal sequence:MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 271)(xx) Nucleic acid sequence of 9F6 VL1 signal sequence:

(SEQ ID NO: 276) ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGT(xxi) Amino acid sequence of 9F6 VL2 and VL3 signal sequence:METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268)(xxii) Nucleic acid sequence of 9F6 VL2 and VL3 signal sequence:

(SEQ ID NO: 273) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(xxiii) Amnio acid sequence of 3G4 VH signal sequence:MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 270)(xxiv) Nucleic acid sequence of 3G4 VH signal sequence:

(SEQ ID NO: 275) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(xxv) Amino acid sequence of 3G4 VL signal sequence:METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268)(xxvi) Nucleic acid sequence of 3G4 VL signal sequence:

(SEQ ID NO: 273) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(xxvii) Amino acid sequence of 17C8 VH signal sequence:MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 270)(xxviii) Nucleic acid sequence of 17C8 VH signal sequence:

(SEQ ID NO: 275) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA(xxix) Amino acid sequence of 17C8 VL signal sequence:METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268)(xxx) Nucleic acid sequence of 17C8 VL signal sequence:

(SEQ ID NO: 273) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA.

In another embodiment, the heavy and light chains of the anti-TIM3antibodies (e.g., TIM3.2 to TIM3.18) can be engineered with signalsequences that differ from those present in the hybridomas from whichthey were cloned. Examples of such sequences include, but not limitedto, the following:

(i) Nucleic acid sequence of signal sequence for the heavy chain:

(SEQ ID NO: 362) ATGAGGGCTTGGATCTTCTTTCTGCTCTGCCTGGCCGGGAGAGCGCTCG CA(ii) Nucleic acid sequence of signal sequence for the light chain:

(SEQ ID NO: 363) ATGAGGGCTTGGATCTTCTTTCTGCTCTGCCTGGCCGGGCGCGCCTTGG CC(iii) Amino acid sequence of signal sequence for the heavy and lightchains: MRAWIFFLLCLAGRALA (SEQ ID NO: 361).

In addition to the antibody chain genes, recombinant expression vectorscan carry regulatory sequences that control the expression of theantibody chain genes in a host cell. The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the antibody chain genes. Such regulatory sequencesare described, for example, in Goeddel (Gene Expression Technology.Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Itwill be appreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences, candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV), SimianVirus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences can beused, such as the ubiquitin promoter or 3-globin promoter. Stillfurther, regulatory elements composed of sequences from differentsources, such as the SRa promoter system, which contains sequences fromthe SV40 early promoter and the long terminal repeat of human T cellleukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol.8:466-472).

In addition to the antibody chain genes and regulatory sequences,recombinant expression vectors can carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Preferred selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like.

Although it is theoretically possible to express the anti-TIM3antibodies described herein in either prokaryotic or eukaryotic hostcells, expression of antibodies in eukaryotic cells, and most preferablymammalian host cells, is the most preferred because such eukaryoticcells, and in particular mammalian cells, are more likely thanprokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today6: 12-13).

Certain mammalian host cells for expressing the recombinant anti-TIM3antibodies described herein include Chinese Hamster Ovary (CHO cells)(including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc.Natl. Acad. Sct. USA 77:4216-4220, used with a DHFR selectable marker,e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.759:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another expression system is the GS geneexpression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841.When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

XII. Assays

Anti-TIM3 antibodies described herein can be tested for binding to humanTIM3 by, for example, standard ELISA. Briefly, microtiter plates arecoated with purified TIM3, and then blocked with bovine serum albumin.Dilutions of antibody (e.g., dilutions of plasma from TIM3-immunizedmice) are added to each well and incubated. The plates are washed andincubated with secondary reagent (e.g., for human antibodies, agoat-anti-human IgG Fc-specific polyclonal reagent) conjugated tohorseradish peroxidase (HRP). After washing, the plates can be developedand analyzed by a spectrophotometer. Sera from immunized mice can thenbe further screened by flow cytometry for binding to a cell lineexpressing human TIM3, but not to a control cell line that does notexpress TIM3. Briefly, the binding of anti-TIM3 antibodies can beassessed by incubating TIM3 expressing CHO cells with the anti-TIM3antibody. The cells can be washed and binding can be detected with ananti-human IgG Ab. Flow cytometric analyses can be performed using aFACScan flow cytometry (Becton Dickinson, San Jose, Calif.). Mice whichdevelop the highest titers can be used for fusions.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with the TIM3 immnunogen. Hybridomas that produce antibodiesthat bind with high affinity to TIM3 can then be subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can then be chosen for makinga cell bank, and for antibody purification.

To purify anti-TIM3 antibodies, selected hybridomas can be grown formonoclonal antibody purification. Supernatants can be filtered andconcentrated before affinity chromatography. Eluted IgG can be checkedby gel electrophoresis and high performance liquid chromatography toensure purity. The buffer solution can be exchanged, and theconcentration can be determined. The monoclonal antibodies can bealiquoted and stored.

To determine if the selected anti-TIM3 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Biotinylated MAb bindingcan be detected with a streptavidin labeled probe. Competition studiesusing unlabeled monoclonal antibodies and biotinylated monoclonalantibodies can be performed using TIM3 coated-ELISA plates as describedabove.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immnunoglobulin overnight at 4° C. After blocking with 1%BSA, the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

To test the binding of monoclonal antibodies to live cells expressingTIM3, flow cytometry can be used, as described in the Examples. Briefly,cell lines expressing membrane-bound TIM3 (grown under standard growthconditions) are mixed with various concentrations of monoclonalantibodies in PB S containing 0.1% BSA at 4° C. for 1 hour. Afterwashing, the cells are reacted with Fluorescein-labeled anti-IgGantibody under the same conditions as the primary antibody staining. Thesamples can be analyzed by FACScan instrument using light and sidescatter properties to gate on single cells and binding of the labeledantibodies is determined. An alternative assay using fluorescencemicroscopy can be used (in addition to or instead of) the flow cytometryassay. Cells can be stained exactly as described above and examined byfluorescence microscopy. This method allows visualization of individualcells, but can have diminished sensitivity depending on the density ofthe antigen.

Anti-TIM3 antibodies can be further tested for reactivity with the TIM3antigen by Western blotting. Briefly, cell extracts from cellsexpressing TIM3 can be prepared and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked with20% mouse serum, and probed with the monoclonal antibodies to be tested.IgG binding can be detected using anti-IgG alkaline phosphatase anddeveloped with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis,Mo.).

Methods for analyzing binding affinity, cross-reactivity, and bindingkinetics of various anti-TIM3 antibodies include standard assays knownin the art, for example, BIACORE™ surface plasmon resonance (SPR)analysis using a BIACORE™ 2000 SPR instrument (Biacore AB, Uppsala,Sweden).

A variety of assays can be used to characterize the biological activityof anti-TIM3 antibodies (which can be used, e.g., for comparingdifferent anti-TIM3 antibodies), such as those described herein:

(1) T cell activation assays, such as assays using purified T cellsobtained from PBMCs of human donors. Assays can be conducted with totalT cells or subpopulations thereof, e.g., Th1 cells, T cytotoxic cells,Treg cells, CD4+ T cells, CD8+ T cells, provided that they express TIM3.Activation may be measured by determining the level of secretion ofcertain cytokines, e.g., interferon-γ or IL-2 or the level ofproliferation of the T cells. Without wanting to be limited to aparticular mechanism of action, binding of TIM3 antibodies to TIM3 on Tcells may prevent binding of TIM3 to a TIM3 ligand (TIM3 putativeligands include Galectin-9, HMGB 1, Semaphorin-4A, CEACAM-1, ILT-4 andphosphatidylserine) and thereby prevent TIM3 mediated signaling in the Tcell thereby preventing negatively regulation of T cells by TIM3.Exemplary assays, including Th1 assays, TIL assays and mixed lymphocytereactions (MLRs) are provided in the Examples;

(2) assays measuring stimulation of macrophages, e.g., M1 or M2macrophage; and

(3) assays measuring secretion of myeloid-associated cytokines, e.g.,TNFα, IL-1β, GM-CSF, IL-6, IL-2, IL-10, CCL2, CCL3, CCL4 or CCL5 fromTIM3 positive myeloid cells. In certain embodiment, anti-TIM3 antibodiesstimulate the secretion of TNFα, IL-1β, GM-CSF, IL-6, and IL-2 and/orinhibit the secretion of IL-10, CCL2, CCL3, CCL4 or CCL5 from TIM3positive myeloid cells.

Generally, any method for testing the biological activity of an agentthat inhibits immune responses can be used to characterize thebiological activity of anti-TIM3 antibodies, e.g., those described inthe literature (including patents and patent applications) relating toTIM3.

XIII. Immunoconjugates, Antibody Derivatives and Diagnostics

Anti-TIM3 antibodies described herein can be used for diagnosticpurposes, including sample testing and in vivo imaging, and for thispurpose the antibody (or binding fragment thereof) can be conjugated toan appropriate detectable agent, to form an immunoconjugate. Fordiagnostic purposes, appropriate agents are detectable labels thatinclude radioisotopes, for whole body imaging, and radioisotopes,enzymes, fluorescent labels and other suitable antibody tags for sampletesting.

The detectable labels that can be linked to any TIM3 antibody describedherein can be any of the various types used currently in the field of invitro diagnostics, including particulate labels including metal solssuch as colloidal gold, isotopes such as I¹²⁵ or Tc⁹⁹ presented forinstance with a peptidic chelating agent of the N₂S₂, N₃S or N₄ type,chromophores including fluorescent markers, luminescent markers,phosphorescent markers and the like, as well as enzyme labels thatconvert a given substrate to a detectable marker, and polynucleotidetags that are revealed following amplification such as by polymerasechain reaction. Suitable enzyme labels include horseradish peroxidase,alkaline phosphatase and the like. For instance, the label can be theenzyme alkaline phosphatase, detected by measuring the presence orformation of chemiluminescence following conversion of 1,2 dioxetanesubstrates such as adamantyl methoxy phosphoryloxy phenyl dioxetane(AMPPD), disodium3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo{3.3.1.13,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and CDP-STAR®or other luminescent substrates well-known to those in the art, forexample the chelates of suitable lanthanides such as Terbium(III) andEuropium(III). The detection means is determined by the chosen label.Appearance of the label or its reaction products can be achieved usingthe naked eye, in the case where the label is particulate andaccumulates at appropriate levels, or using instruments such as aspectrophotometer, a luminometer, a fluorimeter, and the like, all inaccordance with standard practice.

In some embodiments, conjugation methods result in linkages which aresubstantially (or nearly) non-immunogenic, e.g., peptide- (i.e.,amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, andether linkages. These linkages are nearly non-immunogenic and showreasonable stability within serum (see e.g., Senter, P. D., Curr. Opin.Chem. Biol. 13 (2009) 235-244; WO 2009/059278; WO 95/17886).

Depending on the biochemical nature of the moiety and the antibody,different conjugation strategies can be employed. In case the moiety isnaturally occurring or recombinant of between 50 to 500 amino acids,there are standard procedures in text books describing the chemistry forsynthesis of protein conjugates, which can be easily followed by theskilled artisan (see e.g., Hackenberger, C. P. R., and Schwarzer, D.,Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodimentthe reaction of a maleinimido moiety with a cysteine residue within theantibody or the moiety is used. This is an especially suited couplingchemistry in case e.g., a Fab or Fab′-fragment of an antibody is used.Alternatively in one embodiment coupling to the C-terminal end of theantibody or moiety is performed. C-terminal modification of a protein,e.g., of a Fab-fragment, can be performed as described (Sunbul, M. andYin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

In general, site specific reaction and covalent coupling is based ontransforming a natural amino acid into an amino acid with a reactivitywhich is orthogonal to the reactivity of the other functional groupspresent. For example, a specific cysteine within a rare sequence contextcan be enzymatically converted in an aldehyde (see Frese, M. A., andDierks, T., Chem Bio Chem. 10 (2009) 425-427). It is also possible toobtain a desired amino acid modification by utilizing the specificenzymatic reactivity of certain enzymes with a natural amino acid in agiven sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel.17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; andProtease-catalyzed formation of C— N bonds is used by Bordusa, F.,Highlights in Bioorganic Chemistry (2004) 389-403). Site specificreaction and covalent coupling can also be achieved by the selectivereaction of terminal amino acids with appropriate modifying reagents.

The reactivity of an N-terminal cysteine with benzonitrils (see Ren, H.et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used toachieve a site-specific covalent coupling.

Native chemical ligation can also rely on C-terminal cysteine residues(Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology(2009), 22 (Protein Engineering), 65-96).

U.S. Pat. No. 6,437,095 B 1 describes a conjugation method which isbased on the faster reaction of a cysteine within a stretch ofnegatively charged amino acids with a cysteine located in a stretch ofpositively charged amino acids.

The moiety can also be a synthetic peptide or peptide mimic. In case apolypeptide is chemically synthesized, amino acids with orthogonalchemical reactivity can be incorporated during such synthesis (see e.g.,de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since agreat variety of orthogonal functional groups is at stake and can beintroduced into a synthetic peptide, conjugation of such peptide to alinker is standard chemistry.

In order to obtain a mono-labeled polypeptide, the conjugate with 1:1stoichiometry can be separated by chromatography from other conjugationside-products. This procedure can be facilitated by using a dye labeledbinding pair member and a charged linker. By using this kind of labeledand highly negatively charged binding pair member, mono conjugatedpolypeptides are easily separated from non-labeled polypeptides andpolypeptides which carry more than one linker, since the difference incharge and molecular weight can be used for separation. The fluorescentdye can be useful for purifying the complex from unbound components,like a labeled monovalent binder.

In one embodiment the moiety attached to an anti-TIM3 antibody isselected from the group consisting of a binding moiety, a labelingmoiety, and a biologically active moiety.

Anti-TIM3 antibodies described herein can also be conjugated to atherapeutic agent to form an immunoconjugate such as an antibody-drugconjugate (ADC). Suitable therapeutic agents include antimetabolites,alkylating agents, DNA minor groove binders, DNA intercalators, DNAcrosslinkers, histone deacetylase inhibitors, nuclear export inhibitors,proteasome inhibitors, topoisomerase I or II inhibitors, heat shockprotein inhibitors, tyrosine kinase inhibitors, antibiotics, andanti-mitotic agents. In the ADC, the antibody and therapeutic agentpreferably are conjugated via a linker cleavable such as a peptidyl,disulfide, or hydrazone linker. In other embodiments, the linker is apeptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys,Pro-Val-Gly-Val-Val (SEQ ID NO: 300), Ala-Asn-Val, Val-Leu-Lys,Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. The ADCs can beprepared as described in U.S. Pat. Nos. 7,087,600; 6,989,452; and7,129,261; PCT Publications WO 02/096910; WO 07/038658; WO 07/051081; WO07/059404; WO 08/083312; and WO 08/103693; U.S. Patent Publications20060024317; 20060004081; and 20060247295.

Anti-TIM3 antibodies, e.g., those described herein, can also be used fordetecting TIM3, such as human TIM3, e.g., human TIM3 in tissues ortissue samples. The antibodies can be used, e.g., in an ELISA assay orin flow cytometry. In certain embodiments, an anti-TIM3 antibody iscontacted with cells, e.g., cells in a tissue, for a time appropriatefor specific binding to occur, and then a reagent, e.g., an antibodythat detects the anti-TIM3 antibody, is added. Exemplary assays areprovided in the Examples. The anti-TIM3 antibody can be a fully humanantibody, or it can be a chimeric antibody, such as an antibody havinghuman variable regions and murine constant regions or a portion thereof.Exemplary methods for detecting TIM3, e.g., human TIM3, in a sample(cell or tissue sample) comprise (i) contacting a sample with ananti-TIM3 antibody, for a time sufficient for allowing specific bindingof the anti-TIM3 antibody to TIM3 in the sample, and (2) contacting thesample with a detection reagent, e.g., an antibody, that specificallybinds to the anti-TIM3 antibody, such as to the Fc region of theanti-TIM3 antibody, to thereby detect TIM3 bound by the anti-TIM3antibody. Wash steps can be included after the incubation with theantibody and/or detection reagent. Anti-TIM3 antibodies for use in thesemethods do not have to be linked to a label or detection agents, as aseparate detection agent can be used.

Other uses for anti-TIM3 antibodies, e.g., as monotherapy or combinationtherapy, are provided elsewhere herein, e.g., in the section pertainingto combination treatments.

XIV. Bispecific Molecules

Anti-TIM3 antibodies described herein can be used for forming bispecificmolecules. An anti-TIM3 antibody, or antigen-binding portions thereof,can be derivatized or linked to another functional molecule, e.g.,another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. For example, an anti-TIM3antibody can be linked to an antibody or scFv that binds specifically toany protein that can be used as potential targets for combinationtreatments, such as the proteins described herein (e.g., antibodies toPD-1, PD-L1, GITR, or LAG-3). The antibody described herein can in factbe derived or linked to more than one other functional molecule togenerate multispecific molecules that bind to more than two differentbinding sites and/or target molecules; such multispecific molecules arealso intended to be encompassed by the term “bispecific molecule” asused herein. To create a bispecific molecule described herein, anantibody described herein can be functionally linked (e.g., by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other binding molecules, such as another antibody, antibodyfragment, peptide or binding mimetic, such that a bispecific moleculeresults.

Accordingly, provided herein are bispecific molecules comprising atleast one first binding specificity for TIM3 and a second bindingspecificity for a second target epitope. In an embodiment describedherein in which the bispecific molecule is multispecific, the moleculecan further include a third binding specificity.

In one embodiment, the bispecific molecules described herein comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)2, Fv, or a single chainFv (scFv). The antibody can also be a light chain or heavy chain dimer,or any minimal fragment thereof such as a Fv or a single chain constructas described in Ladner et al. U.S. Pat. No. 4,946,778.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules described herein are murine,chimeric and humanized monoclonal antibodies.

The bispecific molecules described herein can be prepared by conjugatingthe constituent binding specificities using methods known in the art.For example, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Some conjugating agents are SATAand sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particular embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, preferably one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,mAb×(scFv)₂, Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecificantibody can comprise an antibody comprising an scFv at the C-terminusof each heavy chain. A bispecific molecule described herein can be asingle chain molecule comprising one single chain antibody and a bindingdeterminant, or a single chain bispecific molecule comprising twobinding determinants. Bispecific molecules can comprise at least twosingle chain molecules. Methods for preparing bispecific molecules aredescribed for example in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175;5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed using art-recognized methods, such as enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay

(RIA), FACS analysis, bioassay (e.g., growth inhibition), or WesternBlot assay. Each of these assays generally detects the presence ofprotein-antibody complexes of particular interest by employing a labeledreagent (e.g., an antibody) specific for the complex of interest.

XV. Compositions

Further provided are compositions, e.g., a pharmaceutical compositions,containing one or a combination of anti-TIM3 antibodies or combinationwith antibodies to other targets, or antigen-binding portion(s) thereof,described herein, formulated together with a pharmaceutically acceptablecarrier. Such compositions can include one or a combination of (e.g.,two or more different) antibodies, or immunoconjugates or bispecificmolecules described herein. For example, a pharmaceutical compositiondescribed herein can comprise a combination of antibodies (orimmunoconjugates or bispecifics) that bind to different epitopes on thetarget antigen or that have complementary activities.

In certain embodiments, a composition comprises an anti-TIM3 antibody ata concentration of at least 1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml, 100mg/ml, 150 mg/ml, 200 mg/ml, 1-300 mg/ml, or 100-300 mg/ml.

Pharmaceutical compositions described herein also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-TIM3 antibody described hereincombined with at least one other anti-cancer and/or immunomodulating,e.g., T-cell stimulating (e.g., activating) agent. Examples oftherapeutic agents that can be used in combination therapy are describedin greater detail below in the section on uses of the anti-TIM3antibodies described herein.

In some embodiments, therapeutic compositions disclosed herein caninclude other compounds, drugs, and/or agents used for the treatment ofcancer. Such compounds, drugs, and/or agents can include, for example,chemotherapy drugs, small molecule drugs or antibodies that stimulatethe immune response to a given cancer. In some instances, therapeuticcompositions can include, for example, one or more of an anti-CTLA-4antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-OX40(also known as CD134, TNFRSF4, ACT35 and/or TXGP1L) antibody, ananti-CD137 antibody, an anti-LAG-3 antibody, an anti-GITR antibody, orany combination thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. In some embodiments, the carrier is suitablefor intravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, or bispecific molecule, can be coated in a material toprotect the compound from the action of acids and other naturalconditions that can inactivate the compound.

The pharmaceutical compounds described herein can include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19).Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydroiodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

A pharmaceutical composition described herein can also include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that can beemployed in the pharmaceutical compositions described herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions can also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms can be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It can also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsdescribed herein is contemplated. A pharmaceutical composition cancomprise a preservative or can be devoid of a preservative.Supplementary active compounds can be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, the compositions caninclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, some methods of preparation are vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, from about 0.1percent to about 70 percent, or from about 1 percent to about 30 percentof active ingredient in combination with a pharmaceutically acceptablecarrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus can beadministered, several divided doses can be administered over time or thedose can be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms described herein are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of an anti-TIM3 antibody, e.g., described herein, thedosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5or 10 mg/kg, of the host body weight. For example dosages can be 0.3mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kgbody weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.An exemplary treatment regime entails administration once per week, onceevery two weeks, once every three weeks, once every four weeks, once amonth, once every 3 months or once every three to 6 months. Exemplarydosage regimens for an anti-TIM3 antibody described herein include 1mg/kg body weight or 3 mg/kg body weight via intravenous administration,with the antibody being given using one of the following dosingschedules: (i) every four weeks for six dosages, then every threemonths; (ii) every three weeks; (iii) 3 mg/kg body weight once followedby 1 mg/kg body weight every three weeks.

An anti-TIM3 antibody can be administered at a flat dose (flat doseregimen). In other embodiments, an anti-TIM3 antibody can beadministered at a fixed dose with another antibody. In certainembodiments, an anti-TIM3 antibody is administered at a dose based onbody weight.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

An anti-TIM3 antibody can be administered with another antibody at thedosage regimen of the other antibody. For example, an anti-TIM3 antibodycan be administered with an anti-PD-1 antibody, such as nivolumab(OPDIVO®), every two weeks as an i.v. infusion over 60 minutes untildisease progression or unacceptable toxicity occurs. An anti-TIM3antibody can be administered with pembrolizumab (KEYTRUDA®) every 3weeks as an i.v. infusion over 30 minutes until disease progression orunacceptable toxicity occurs. An anti-TIM3 antibody can be administeredwith atezolizumab (TECENTRIQ™) every 3 weeks as an i.v. infusion over 60or 30 minutes until disease progression or unacceptable toxicity occurs.

An antibody can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage andfrequency vary depending on the half-life of the antibody in thepatient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions described herein can be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions described herein employed,or the ester, salt or amide thereof, the route of administration, thetime of administration, the rate of excretion of the particular compoundbeing employed, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular compositionsemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and like factors wellknown in the medical arts.

A “therapeutically effective dosage” of an anti-TIM3 antibody describedherein can result in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction. Inthe context of cancer, a therapeutically effective dose can result inincreased survival, e.g., overall survival, and/or prevention of furtherdeterioration of physical symptoms associated with cancer. Symptoms ofcancer are well-known in the art and include, for example, unusual molefeatures, a change in the appearance of a mole, including asymmetry,border, color and/or diameter, a newly pigmented skin area, an abnormalmole, darkened area under nail, breast lumps, nipple changes, breastcysts, breast pain, death, weight loss, weakness, excessive fatigue,difficulty eating, loss of appetite, chronic cough, worseningbreathlessness, coughing up blood, blood in the urine, blood in stool,nausea, vomiting, liver metastases, lung metastases, bone metastases,abdominal fullness, bloating, fluid in peritoneal cavity, vaginalbleeding, constipation, abdominal distension, perforation of colon,acute peritonitis (infection, fever, pain), pain, vomiting blood, heavysweating, fever, high blood pressure, anemia, diarrhea, jaundice,dizziness, chills, muscle spasms, colon metastases, lung metastases,bladder metastases, liver metastases, bone metastases, kidneymetastases, and pancreatic metastases, difficulty swallowing, and thelike.

A therapeutically effective dose can prevent or delay onset of cancer,such as can be desired when early or preliminary signs of the diseaseare present. Laboratory tests utilized in the diagnosis of cancerinclude chemistries (including the measurement of TIM3 levels),hematology, serology and radiology. Accordingly, any clinical orbiochemical assay that monitors any of the foregoing can be used todetermine whether a particular treatment is a therapeutically effectivedose for treating cancer. One of ordinary skill in the art would be ableto determine such amounts based on such factors as the subject's size,the severity of the subject's symptoms, and the particular compositionor route of administration selected.

A composition described herein can be administered via one or moreroutes of administration using one or more of a variety of methods knownin the art. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. Routes of administration for the anti-TIM3 antibodies describedherein can include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, an antibody described herein could potentially beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a particular embodiment, a therapeuticcomposition described herein can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules for use withanti-TIM3 antibodies described herein include: U.S. Pat. No. 4,487,603,which discloses an implantable micro-infusion pump for dispensingmedication at a controlled rate; U.S. Pat. No. 4,486,194, whichdiscloses a therapeutic device for administering medicaments through theskin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the anti-TIM3 antibodies described herein can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds described herein cross the BBB (ifdesired, e.g., for brain cancers), they can be formulated, for example,in liposomes. For methods of manufacturing liposomes, see, e.g., U.S.Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes cancomprise one or more moieties which are selectively transported intospecific cells or organs, thus enhance targeted drug delivery (see,e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplarytargeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem.Blophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman et al.(1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. AgentsChemother. 39: 180); surfactant protein A receptor (Briscoe et al.(1995) Am. J. Physiol. 1233: 134); p¹²⁰ (Schreier et al. (1994) J. Biol.Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.346: 123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

XVI. Uses and Methods

The antibodies, antibody compositions and methods described herein havenumerous in vitro and in vivo utilities involving, for example,enhancement of immune response, such as by inhibiting (or antagonizing)TIM3 (e.g., signaling), or detection of TIM3. In one embodiment, theanti-TIM3 antibodies described herein are human antibodies. For example,anti-TIM3 antibodies described herein can be administered to cells inculture, in vitro or ex vivo, or to human subjects, e.g., in vivo, toenhance immunity in a variety of diseases. Accordingly, provided hereinare methods of modifying an immune response in a subject comprisingadministering to the subject an anti-TIM3 antibody, or antigen-bindingportion thereof, described herein such that the immune response in thesubject is modified. In some embodiments, the response is enhanced,stimulated or up-regulated.

Subjects suitable for the present methods include human patients in whomenhancement of an immune response would be desirable. The methods areparticularly suitable for treating human patients having a disorder thatcan be treated by augmenting an immune response (e.g., a T-cell mediatedimmune response, e.g., an antigen specific T cell response). In aparticular embodiment, the methods are particularly suitable fortreatment of cancer in vivo. To achieve antigen-specific enhancement ofimmunity, anti-TIM3 antibodies described herein can be administeredtogether with an antigen of interest or the antigen can already bepresent in the subject to be treated (e.g., a tumor-bearing orvirus-bearing subject). When antibodies to TIM3 are administeredtogether with another agent, the two can be administered separately orsimultaneously.

Also encompassed are methods for detecting the presence of human TIM3antigen in a sample, or measuring the amount of human TIM3 antigen,comprising contacting the sample, and a control sample, with amonoclonal antibody, e.g., a human monoclonal antibody, or an antigenbinding portion thereof, which specifically binds to human TIM3, underconditions that allow for formation of a complex between the antibody orportion thereof and human TIM3. The formation of a complex is thendetected, wherein a difference complex formation between the samplecompared to the control sample is indicative the presence of human TIM3antigen in the sample. Moreover, the anti-TIM3 antibodies describedherein can be used to purify human TIM3 via immunoaffinity purification.

Given the ability of anti-TIM3 antibodies described herein to stimulateor co-stimulate T cell responses, e.g., antigen-specific T cellresponses, such as by inhibiting negative effects of TIM3, providedherein are in vitro and in vivo methods of using the anti-TIM3antibodies described herein to stimulate, enhance or upregulateantigen-specific T cell responses, e.g., anti-tumor T cell responses. Incertain embodiments, CD3 stimulation is also provided (e.g., bycoincubation with a cell expressing membrane CD3), which stimulation canbe provided at the same time, before, or after stimulation with ananti-TIM3 antibody. For example, provided herein are methods ofstimulating an antigen-specific T cell response comprising contactingsaid T cell with an anti-TIM3 antibody described herein, and optionallywith an anti-CD3 antibody, such that an antigen-specific T cell responseis stimulated.

Any suitable indicator of an antigen-specific T cell response can beused to measure the antigen-specific T cell response. Non-limitingexamples of such suitable indicators include increased T cellproliferation in the presence of the antibody and/or increase cytokineproduction in the presence of the antibody. In some embodiments,interleukin-2 and/or interferon-γ production by the antigen-specific Tcell is stimulated.

T cells that can be enhanced or co-stimulated with anti-TIM3 antibodiesinclude CD4+ T cells and CD8+ T cells. The T cells can be Teff cells,e.g., CD4+ Teff cells, CD8+ Teff cells, Thelper (Th) cells (e.g., Th1cells) or T cytotoxic (Tc) cells.

Further encompassed are methods of stimulating an immune response (e.g.,an antigen-specific T cell response) in a subject comprisingadministering an anti-TIM3 antibody described herein to the subject suchthat an immune response (e.g., an antigen-specific T cell response) inthe subject is stimulated. In some embodiments, the subject is atumor-bearing subject and an immune response against the tumor isstimulated. A tumor can be a solid tumor or a liquid tumor, e.g., ahematological malignancy. In certain embodiments, a tumor is animmunogenic tumor. In certain embodiments, a tumor is non-immunogenic.In certain embodiments, a tumor is PD-L1 positive. In certainembodiments a tumor is PD-L1 negative. A subject can also be avirus-bearing subject and an immune response against the virus isstimulated.

Further provided are methods for inhibiting growth of tumor cells in asubject comprising administering to the subject an anti-TIM3 antibodydescribed herein such that growth of the tumor is inhibited in thesubject. Also provided are methods of treating a viral infection in asubject comprising administering to the subject an anti-TIM3 antibodydescribed herein such that the viral infection is treated in thesubject.

In certain embodiments, an anti-TIM3 antibody is given to a subject asan adjunctive therapy. Treatments of subjects having cancer with ananti-TIM3 antibody can lead to prolonged survival, e.g., long-termdurable response relative to the current standard of care; long termsurvival of at least 3 months, 6 months, 9 months, 1, 2, 3, 4, 5, 10 ormore years, or recurrence-free survival of at least 3 months, 6 months,9 months, 1, 2, 3, 4, 5, or 10 or more years. In certain embodiments,treatment of a subject having cancer with an anti-TIM3 antibody preventsrecurrence of cancer or delays recurrence of cancer by, e.g., 3 months,6 months, 9 months, 1, 2, 3, 4, 5, or 10 or more years. An anti-TIM3treatment can be used as a first-, second-, or third-line treatment.

Treatment of a subject having cancer with an anti-TIM3 antibodydescribed herein, e.g., TIM3.18.IgG1, can result in, e.g., stabledisease, partial response, increased overall survival, increased diseasefree survival, or enhanced progression free survival.

In certain embodiments, an anti-TIM3 antibody described herein is notsignificantly toxic. For example, a TIM3 antibody is not significantlytoxic to an organ of a human, e.g., one or more of the liver, kidney,brain, lungs, and heart, as determined, e.g., in clinical trials. Incertain embodiments, a TIM3 antibody does not significantly trigger anundesirable immune response, e.g., autoimmunity or inflammation.

In certain embodiments, treatment of a subject with an anti-TIM3antagonist (e.g., an anti-TIM3 antibody described herein) does notresult in overstimulation of the immune system to the extent that thesubject's immune system then attacks the subject itself (e.g.,autoimmune response) or results in, e.g., anaphylaxis. Thus, in someembodiments, anti-TIM3 antibodies do not cause anaphylaxis.

In certain embodiments, treatment of a subject with an anti-TIM3antibody described herein, e.g., an antibody comprising the CDRs orvariable regions of 13A3 or a variant thereof (e.g., as describedherein) or other anti-TIM3 antibodies described herein, does not causesignificant inflammatory reactions, e.g., immune-mediated pneumonitis,immune-mediated colitis, immune mediated hepatitis, immune-mediatednephritis or renal dysfunction, immune-mediated hypophysitis,immune-mediated hypothyroidism and hyperthyroidism, or otherimmune-mediated adverse reactions. In certain embodiments, an anti-TIM3antibody comprising the CDRs or variable regions of 13A3 or a variantthereof (e.g., as described herein) causes fewer inflammatory reactions,e.g., immune-mediated pneumonitis, immune-mediated colitis, immunemediated hepatitis, immune-mediated nephritis or renal dysfunction,immune-mediated hypophysitis, immune-mediated hypothyroidism andhyperthyroidism, anaphylaxis or other immune-mediated adverse reactions,than other anti-TIM3 antibodies. In certain embodiments, treatment of asubject with an anti-TIM3 antibody described herein, e.g., an antibodycomprising the CDRs or variable regions of 13A3 or a variant thereof(e.g., as described herein) or other anti-TIM3 antibodies describedherein, does not cause significant cardiac disorders, e.g., ventriculararrhythmia; eye disorders, e.g., iridocyclitis; infusion-relatedreactions; increased amylase, increased lipase; nervous systemdisorders, e.g., dizziness, peripheral and sensory neuropathy; skin andsubcutaneous tissue disorders, e.g., rash, pruritus, exfoliativedermatitis, erythema multiforme, vitiligo or psoriasis; respiratory,thoracic and mediastinal disorders, e.g., cough; fatigue; nausea;decreased appetite; constipation; arthralgia; or diarrhea.

In certain embodiments, an anti-TIM3 antibody provides synergisticanti-tumor effects in combination with another cancer therapy, such as acompound that stimulates the immune system (e.g., an immuno-oncologyagent), e.g., a compound described herein or a compound modulating atarget described herein.

Using human antibodies, as opposed to chimeric or humanized antibodies,may result in a lower levels of anti-drug antibodies (ADA). Accordingly,the human anti-TIM3 antibodies described herein, e.g., TIM3.18.IgG1.3,may have lower ADA relative to anti-TIM3 antibodies that are not humanantibodies (e.g., relative to humanized or chimeric anti-TIM3antibodies).

These and other methods described herein are discussed in further detailbelow.

XVI.A. Cancer

Inhibition of TIM3 by anti-TIM3 antibodies can enhance the immuneresponse to cancerous cells in a patient having cancer. Provided hereinare methods for treating a subject having cancer, comprisingadministering to the subject an anti-TIM3 antibody described herein,such that the subject is treated, e.g., such that growth of canceroustumors is inhibited or reduced and/or that the tumors regress and/orthat prolonged survival is achieved. An anti-TIM3 antibody can be usedalone to inhibit the growth of cancerous tumors. Alternatively, ananti-TIM3 antibody can be used in conjunction with another agent, e.g.,another immunogenic agent, a standard cancer treatment, or anotherantibody, as described below.

Accordingly, provided herein are methods of treating cancer, e.g., byinhibiting growth of tumor cells, in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-TIM3antibody described herein, e.g., TIM3.2, TIM3.4, TIM3.5, TIM3.6, 9F6,8B9, TIM3.9, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15,TIM3.16, TIM3.17, TIM3.18, TIM3.7, and TIM3.8 having a wildtype IgGconstant region or a constant region having reduced effector function,e.g., IgG1.1 or IgG1.3, or antigen-binding portion thereof. The antibodycan be a human anti-TIM3 antibody (such as any of the human anti-humanTIM3 antibodies described herein). Cancers whose growth can be inhibitedusing the antibodies of the disclosure include cancers typicallyresponsive to immunotherapy and those that are not typically responsiveto immunotherapy. Cancers that can be treated also include TIM3 positivecancers. Cancers can be cancers with solid tumors or blood malignancies(liquid tumors). Non-limiting examples of cancers for treatment includesquamous cell carcinoma, small-cell lung cancer, non-small cell lungcancer, squamous non-small cell lung cancer (NSCLC), nonsquamous NSCLC,glioma, gastrointestinal cancer, renal cancer (e.g., clear cellcarcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrialcancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostatecancer (e.g., hormone refractory prostate adenocarcinoma), thyroidcancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastomamultiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma,breast cancer, colon carcinoma, and head and neck cancer (or carcinoma),gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal naturalkiller, melanoma (e.g., metastatic malignant melanoma, such as cutaneousor intraocular malignant melanoma), bone cancer, skin cancer, uterinecancer, cancer of the anal region, testicular cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,solid tumors of childhood, cancer of the ureter, carcinoma of the renalpelvis, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer,squamous cell cancer, T-cell lymphoma, environmentally-induced cancersincluding those induced by asbestos, vims-related cancers or cancers ofviral origin (e.g., human papilloma vims (HPV-related or -originatingtumors)), and hematologic malignancies derived from either of the twomajor blood cell lineages, i.e., the myeloid cell line (which producesgranulocytes, erythrocytes, thrombocytes, macrophages and mast cells) orlymphoid cell line (which produces B, T, NK and plasma cells), such asall types of leukemias, lymphomas, and myelomas, e.g., acute, chronic,lymphocytic and/or myelogenous leukemias, such as acute leukemia (ALL),acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL),and chronic myelogenous leukemia (CML), undifferentiated AML (MO),myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellmaturation), promyelocytic leukemia (M3 or M3 variant [M3V]),myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]),monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia(M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such asHodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B cellhematologic malignancy, e.g., B-cell lymphomas, T-cell lymphomas,lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma,mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1+) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle celllymphoma, angio immunoblastic T-cell lymphoma, angiocentric lymphoma,intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma,precursor T-lymphoblastic lymphoma, T-lymphoblastic; andlymphoma/leukaemia (T-Lbly/T-ALL), peripheral T-cell lymphoma,lymphoblastic lymphoma, post-transplantation lymphoproliferativedisorder, true histiocytic lymphoma, primary central nervous systemlymphoma, primary effusion lymphoma, B cell lymphoma, lymphoblasticlymphoma (LBL), hematopoietic tumors of lymphoid lineage, acutelymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt'slymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL),immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides orSezary syndrome), and lymphoplasmacytoid lymphoma (LPL) withWaldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, lightchain myeloma, nonsecretory myeloma, smoldering myeloma (also calledindolent myeloma), solitary plasmocytoma, and multiple myelomas, chroniclymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors ofmyeloid lineage, tumors of mesenchymal origin, including fibrosarcomaand rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the centraland peripheral nervous, including astrocytoma, schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer andteratocarcinoma, hematopoietic tumors of lymphoid lineage, for exampleT-cell and B-cell tumors, including but not limited to T-cell disorderssuch as T-prolymphocytic leukemia (T-PLL), including of the small celland cerebriform cell type; large granular lymphocyte leukemia (LGL) ofthe T-cell type; a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head orneck, renal cancer, rectal cancer, cancer of the thyroid gland; acutemyeloid lymphoma, as well as any combinations of said cancers. Themethods described herein can also be used for treatment of metastaticcancers, unresectable, refractory cancers (e.g., cancers refractory toprevious immunotherapy, e.g., with a blocking CTLA-4 or PD-1 antibody),and/or recurrent cancers.

In certain embodiments, an anti-TIM3 antibody is administered topatients having a cancer that exhibited an inadequate response to, orprogressed on, a prior treatment, e.g., a prior treatment with animmuno-oncology or immunotherapy drug, or patients having a cancer thatis refractory or resistant, either intrinsically refractory or resistant(e.g., refractory to a PD-1 pathway antagonist), or a wherein theresistance or refractory state is acquired. For example, subjects whoare not responsive or not sufficiently responsive to a first therapy orwho see disease progression following treatment, e.g., anti-PD-1treatment, can be treated by administration of an anti-TIM3 antibodyalone or in combination with another therapy (e.g., with an anti-PD-1therapy).

In certain embodiments, an anti-TIM3 antibody is administered topatients who have not previously received (i.e., been treated with) animmuno-oncology agent, e.g., a PD-1 pathway antagonist.

In certain embodiments, a method of treating cancer in a subjectcomprises first determining whether the subject is TIM3 positive, e.g.,has tumor cells or TILs that express TIM3, and if the subject has TIM3positive cancer or TIL cells, then administering to the subject ananti-TIM3 antibody, e.g., described herein. A method of treating asubject having cancer with an anti-TIM3 antibody may compriseadministering to a subject who has cancer cells or TIL cells thatexpress TIM3, a therapeutically effective amount of a TIM3 antibody.Also provided herein are methods for predicting whether a subject willrespond to treatment with an anti-TIM3 antibody, wherein the methodscomprise determining the level of TIM3 in cancer or TIL cells of thepatient, and if cancer or TIL cells of the subject are TIM3 positive,then the subject is likely to respond to a treatment with a TIM3antibody.

In certain embodiments, a method of treating cancer in a subjectcomprises first determining whether the subject is PD-L1 or PD-1positive, e.g., has tumor cells or TILs that express PD-L1 or PD-1, andif the subject has PD-L1 or PD-1 positive cancer or TIL cells, thenadministering to the subject an anti-TIM3 antibody (and optionally aPD-1 or PD-L1 antagonist), e.g., described herein. A method of treatinga subject having cancer with an anti-TIM3 antibody (and optionally aPD-1 or PD-L1 antagonist) may comprise administering to a subject whohas cancer cells or TIL cells that express PD-L1 or PD-1, atherapeutically effective amount of a TIM3 antibody (and optionally aPD-1 or PD-L1 antagonist). Also provided herein are methods forpredicting whether a subject will respond to treatment with an anti-TIM3antibody (and optionally a PD-1 or PD-L1 antagonist), wherein themethods comprise determining the level of PD-L1 or PD-1 in cancer or TILcells of the patient, and if cancer or TIL cells of the subject arePD-L1 or PD-1 positive, then the subject is likely to respond to atreatment with a TIM3 antibody (and optionally a PD-1 or PD-L1antagonist).

An anti-TIM3 antibody can be administered with a standard of caretreatment. An anti-TIM3 antibody can be administered as a maintenancetherapy, e.g., a therapy that is intended to prevent the occurrence orrecurrence of tumors.

An anti-TIM3 antibody can be administered with another treatment, e.g.,radiation, surgery, or chemotherapy. For example e, anti-TIM3 antibodyadjunctive therapy can be administered when there is a risk thatmicrometastases can be present and/or in order to reduce the risk of arelapse.

An anti-TIM3 antibody can be administered as a monotherapy, or as theonly immuno stimulating therapy. Antibodies to TIM3, e.g., theanti-TIM3, can also be combined with an immunogenic agent, such ascancerous cells, purified tumor antigens (including recombinantproteins, peptides, and carbohydrate molecules), cells, and cellstransfected with genes encoding immune stimulating cytokines (He et al.,(2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccinesthat can be used include peptides of melanoma antigens, such as peptidesof gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cellstransfected to express the cytokine GM-CSF (discussed further below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. By lowering the threshold of T cell activation via TIM3inhibition, the tumor responses in the host can be activated, allowingtreatment of non-immunogenic tumors or those having limitedimmunogenicity.

An anti-TIM3 antibody, e.g., an anti-TIM3 antibody described herein, canbe combined with a vaccination protocol. Many experimental strategiesfor vaccination against tumors have been devised (see Rosenberg, S.,2000, Development of Cancer Vaccines, ASCO Educational Book Spring:60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302;Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000,ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol,M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.),1997, Cancer: Principles and Practice of Oncology, Fifth Edition). Inone of these strategies, a vaccine is prepared using autologous orallogeneic tumor cells. These cellular vaccines have been shown to bemost effective when the tumor cells are transduced to express GM-CSF.GM-CSF has been shown to be a potent activator of antigen presentationfor tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. SciU.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. TIM3 inhibition can be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen can include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al. (1994) Science266: 2011-2013). Tumor antigen can also be “neo-antigens” expressed incancer cells because of somatic mutations that alter protein sequence orcreate fusion proteins between two unrelated sequences (i.e., bcr-abl inthe Philadelphia chromosome), or idiotype from B cell tumors.

Other tumor vaccines can include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which can be used in conjunction with TIM3inhibition is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot & Srivastava(1995) Science 269: 1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DCs can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withTIM3 inhibition to activate more potent anti-tumor responses.

TIM3 inhibition can also be combined with standard cancer treatments(e.g., surgery, radiation, and chemotherapy). TIM3 inhibition can beeffectively combined with chemotherapeutic regimes. In these instances,it can be possible to reduce the dose of chemotherapeutic reagentadministered (Mokyr et al. (1998) Cancer Research 58: 5301-5304). Anexample of such a combination is an anti-TIM3 antibody in combinationwith decarbazine for the treatment of melanoma. Another example of sucha combination is an anti-TIM3 antibody in combination with interleukin-2(IL-2) for the treatment of melanoma. The scientific rationale behindthe combined use of TIM3 inhibition and chemotherapy is that cell death,that is a consequence of the cytotoxic action of most chemotherapeuticcompounds, should result in increased levels of tumor antigen in theantigen presentation pathway. Other combination therapies that canresult in synergy with TIM3 inhibition through cell death are radiation,surgery, and hormone deprivation. Each of these protocols creates asource of tumor antigen in the host. Angiogenesis inhibitors can also becombined with TIM3 inhibition. Inhibition of angiogenesis leads to tumorcell death which can feed tumor antigen into host antigen presentationpathways.

The anti-TIM3 antibodies described herein can also be used incombination with bispecific antibodies that target Fca or Fcγreceptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used totarget two separate antigens. For example anti-Fc receptor/anti tumorantigen (e.g., Her-2/neu) bispecific antibodies have been used to targetmacrophages to sites of tumor. This targeting can more effectivelyactivate tumor specific responses. The T cell arm of these responseswould be augmented by the inhibition of TIM3. Alternatively, antigen canbe delivered directly to DCs by the use of bispecific antibodies whichbind to tumor antigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms can be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-β (Kehrl et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365).Antibodies to each of these entities can be used in combination withanti-TIM3 antibodies to counteract the effects of the immunosuppressiveagent and favor tumor immune responses by the host.

Other antibodies which activate host immune responsiveness can be usedin combination with anti-TIM3 antibodies. These include molecules on thesurface of dendritic cells which activate DC function and antigenpresentation. Anti-CD40 antibodies are able to substitute effectivelyfor T cell helper activity (Ridge et al. (1998) Nature 393: 474-478) andcan be used in conjunction with anti-TIM3 antibodies. Activatingantibodies to T cell costimulatory molecules such as CTLA-4 (e.g., U.S.Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164:2160-2169), 4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685(1997), and ICOS (Hutloff et al. (1999) Nature 397: 262-266) can alsoprovide for increased levels of T cell activation. Inhibitors of PD 1 orPD-L1 can also be used in conjunction with an anti-TIM3 antibody. Othercombination are provided elsewhere herein.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit can be obtained fromgraft vs. tumor responses. TIM3 inhibition can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to stimulateantigen-specific T cells against tumor (Greenberg & Riddell (1999)Science 285: 546-51). These methods can also be used to activate T cellresponses to infectious agents such as CMV. Ex vivo activation in thepresence of anti-TIM3 antibodies can increase the frequency and activityof the adoptively transferred T cells.

XVI.B. Infectious Diseases

Methods described herein can also be used to treat patients that havebeen exposed to particular toxins or pathogens. Accordingly, anotheraspect described herein provides a method of treating an infectiousdisease in a subject comprising administering to the subject ananti-TIM3 antibody, or antigen-binding portion thereof, such that thesubject is treated for the infectious disease. Additionally oralternatively, the antibody can be a chimeric or humanized antibody.

Similar to its application to tumors as discussed above,antibody-mediated TIM3 inhibition can be used alone, or as an adjuvant,in combination with vaccines, to stimulate the immune response topathogens, toxins, and self-antigens. Examples of pathogens for whichthis therapeutic approach can be particularly useful, include pathogensfor which there is currently no effective vaccine, or pathogens forwhich conventional vaccines are less than completely effective. Theseinclude, but are not limited to HIV, Hepatitis (A, B, & C), Influenza,Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonasaeruginosa. TIM3 inhibition can be useful against established infectionsby agents such as HIV that present altered antigens over the course ofthe infections. These novel epitopes are recognized as foreign at thetime of anti-human TIM3 antibody administration, thus provoking a strongT cell response.

Some examples of pathogenic viruses causing infections treatable bymethods described herein include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr vims),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella vims, parvovirus, vaccinia virus, HTLVvirus, dengue vims, papillomavirus, molluscum virus, poliovirus, rabiesvirus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods described herein include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods described herein include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods described herein include Entamoeba histolytica, Balantidum coli,Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp.,Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosomabrucel, Trypanosoma cruzi, Leishmania donovant, Toxoplasma gondii, andNippostrongylus brasiliensis.

In all of the above methods, TIM3 inhibition can be combined with otherforms of immunotherapy, e.g., those described herein, such as cytokinetreatment (e.g., interferons, GM-CSF, G-CSF, IL-2), or bispecificantibody therapy, which provides for enhanced presentation of tumorantigens (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak (1994) Structure 2: 1121-1123).

XVI.C. Autoimmune Reactions

Anti-TIM3 antibodies could provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many antitumor responses involve anti-selfreactivities (vanElsas et al. (2001) J. Exp. Med. 194:481-489; Overwijk,et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987; Hurwitz,(2000) supra; Rosenberg & White (1996) J. Immunother Emphasis TumorImmunol 19 (1): 81-4). Therefore, it is possible to consider usinganti-TIM3 antibodies in conjunction with various self proteins in orderto devise vaccination protocols to efficiently generate immune responsesagainst these self proteins for disease treatment. For example,Alzheimer's disease involves inappropriate accumulation of AB peptide inamyloid deposits in the brain; antibody responses against amyloid areable to clear these amyloid deposits (Schenk et al., (1999) Nature 400:173-177).

Other self proteins can also be used as targets such as IgE for thetreatment of allergy and asthma, and TNF-α for rheumatoid arthritis.Finally, antibody responses to various hormones can be induced by theuse of anti-TIM3 antibodies. Neutralizing antibody responses toreproductive hormones can be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors can also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-TIM3 antibodiescan be used for induction of therapeutic autoimmune responses to treatpatients having an inappropriate accumulation of other self-antigens,such as amyloid deposits, including AB in Alzheimer's disease, cytokinessuch as TNF-α, and IgE.

XVI.D. Vaccines

Anti-TIM3 antibodies described herein can be used to stimulateantigen-specific immune responses by co-administration of an anti-TIM3antibody with an antigen of interest (e.g., a vaccine). Accordingly,provided herein are methods of enhancing an immune response to anantigen in a subject, comprising administering to the subject: (i) theantigen; and (ii) an anti-TIM3 antibody, or antigen-binding portionthereof, such that an immune response to the antigen in the subject isenhanced. The antibody can be a human anti-human TIM3 antibody (such asany of the human anti-TIM3 antibodies described herein). Additionally oralternatively, the antibody can be a chimeric or humanized antibody. Theantigen can be, for example, a tumor antigen, a viral antigen, abacterial antigen or an antigen from a pathogen. Non-limiting examplesof such antigens include those discussed in the sections above, such asthe tumor antigens (or tumor vaccines) discussed above, or antigens fromthe viruses, bacteria or other pathogens described above.

In certain embodiments, a peptide or fusion protein comprising theepitope to which an anti-TIM3 antibody binds is used as a vaccineinstead of, or in addition to, an anti-TIM3 antibody.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) described herein in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, anti-TIM3 antibodies described herein can beco-administered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immuno-complex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be coadministered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, bythemselves, are only effective at levels which are toxic or subtoxic toa patient. Cisplatin is intravenously administered as a 100 mg/ml doseonce every four weeks and adriamycin is intravenously administered as a60-75 mg/ml dose once every 21 days. Co-administration of anti-TIM3antibodies, or antigen binding fragments thereof, described herein withchemotherapeutic agents provides two anti-cancer agents which operatevia different mechanisms which yield a cytotoxic effect to human tumorcells. Such coadministration can solve problems due to development ofresistance to drugs or a change in the antigenicity of the tumor cellswhich would render them unreactive with the antibody.

Also within the scope described herein are kits comprising the antibodycompositions described herein (e.g., human antibodies, bispecific ormultispecific molecules, or immunoconjugates) and instructions for use.The kit can further contain at least one additional reagent, or one ormore additional human anti-TIM3 antibodies described herein (e.g., ahuman antibody having a complementary activity which binds to an epitopein TIM3 antigen distinct from the first human antibody). Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit.

XVI.E. Combination Therapies

In addition to the combinations therapies provided above, anti-TIM3antibodies, e.g., those described herein, can also be used incombination therapy, e.g., for treating cancer, as described below.

Provided herein are methods of combination therapy in which an anti-TIM3antibody is coadministered with one or more additional agents, e.g.,small molecule drugs, antibodies or antigen binding portions thereof,that are effective in stimulating immune responses to thereby furtherenhance, stimulate or upregulate immune responses in a subject.

Generally, an anti-TIM3 antibody, e.g., described herein, can becombined with (i) an agonist of a stimulatory (e.g., co-stimulatory)molecule (e.g., receptor or ligand) and/or (ii) an antagonist of aninhibitory signal or molecule (e.g., receptor or ligand) on immunecells, such as T cells, both of which result in amplifying immuneresponses, such as antigen-specific T cell responses. In certainaspects, an immuno-oncology agent is (i) an agonist of a stimulatory(including a co-stimulatory) molecule (e.g., receptor or ligand) or (ii)an antagonist of an inhibitory (including a co-inhibitory) molecule(e.g., receptor or ligand) on cells, e.g., those inhibiting T cellactivation or those involved in innate immunity, e.g., NK cells, andwherein the immuno-oncology agent enhances innate immunity. Suchimmuno-oncology agents are often referred to as immune checkpointregulators, e.g., immune checkpoint inhibitor or immune checkpointstimulator.

In certain embodiments, an anti-TIM3 antibody is administered with anagent that targets a stimulatory or inhibitory molecule that is a memberof the immunoglobulin super family (IgSF). For example, anti-TIM3antibodies, e.g., described herein, can be administered to a subjectwith an agent that targets a member of the IgSF family to increase animmune response. For example, an anti-TIM3 antibody can be administeredwith an agent that targets (or binds specifically to) a member of the B7family of membrane-bound ligands that includes B7-1, B7-2, B7-H1(PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), andB7-H6 or a co-stimulatory or co-inhibitory receptor or ligand bindingspecifically to a B7 family member.

An anti-TIM3 antibody can also be administered with an agent thattargets a member of the TNF and TNFR family of molecules (ligands orreceptors), such as CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30,CD30L, 4-1BBL, CD137, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3,TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn 14, TWEAK, BAFFR, EDAR, XEDAR,TACI, APRIL, BCMA, LTpR, LIGHT, DcR3, HVEM, VEGI/TL 1A, TRAMP/DR3, EDA1,EDA2, TNFR1, Lymphotoxin a/TNFp, TNFR2, TNFa, LTpR, Lymphotoxin a 1β2,FAS, FASL, RELT, DR6, TROY, and NGFR (see, e.g., Tansey (2009) DrugDiscovery Today 00: 1).

T cell responses can be stimulated by a combination of anti-TIM3antibodies having the variable regions of, e.g., TIM3.2, TIM3.4, TIM3.5,TIM3.6, 9F6, 8B9, TIM3.9, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14,TIM3.15, TIM3.16, TIM3.17, TIM3.18, TIM3.7, and TIM3.8, and one or moreof the following agents:

(1) An antagonist (inhibitor or blocking agent) of a protein thatinhibits T cell activation (e.g., immune checkpoint inhibitors), such asCTLA-4, PD-1, PD-L1, PD-L2, GITR, and LAG-3, Galectin 9, CEACAM-1, BTLA,CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48,GARP, PD1H, LAIR1, TIM-1, and TIM-4; and/or

(2) An agonist of a protein that stimulates T cell activation, such asB7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, GITR, ICOS, ICOS-L, OX40,OX40L, CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and can becombined with anti-TIM3 antibodies, e.g., those described herein, fortreating cancer, include: YERVOY® (ipilimumab) or Tremelimumab (toCTLA-4), galiximab (to B7.1), BMS-936558 (to PD-1), MK-3475 (to PD-1),atezolizumab (TECENTRIQ®), AMP224 (to B7DC), BMS-936559 (to B7-H1),MPDL3280A (to B7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (toB7H3), IMP321 (to LAG-3), BMS-663513 (to CD137), PF-05082566 (to CD137),CDX-1127 (to CD27), anti-OX40 (Providence Health Services), huMAbOX40L(to OX40L), Atacicept (to TACI), CP-870893 (to CD40), Lucatumumab (toCD40), Dacetuzumab (to CD40), Muromonab-CD3 (to CD3); anti-GITRantibodies MK4166, TRX518, Medi1873, INBRX-110, LK2-145, GWN-323,GITRL-Fc, or any combination thereof.

Other molecules that can be combined with anti-TIM3 antibodies for thetreatment of cancer include antagonists of inhibitory receptors on NKcells or agonists of activating receptors on NK cells. For example,anti-TIM3 antibodies can be combined with antagonists of KIR (e.g.,lirilumab).

T cell activation is also regulated by soluble cytokines, and anti-TIM3antibodies can be administered to a subject, e.g., having cancer, withantagonists of cytokines that inhibit T cell activation or agonists ofcytokines that stimulate T cell activation.

In certain embodiments, anti-TIM3 antibodies can be used in combinationwith (i) antagonists (or inhibitors or blocking agents) of proteins ofthe IgSF family or B7 family or the TNF family that inhibit T cellactivation or antagonists of cytokines that inhibit T cell activation(e.g., IL-6, IL-10, TGF-β, VEGF; “immunosuppressive cytokines”) and/or(ii) agonists of stimulatory receptors of the IgSF family, B7 family orthe TNF family or of cytokines that stimulate T cell activation, forstimulating an immune response, e.g., for treating proliferativediseases, such as cancer.

Yet other agents for combination therapies include agents that inhibitor deplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155(WO11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716,WO13/132044) or FPA-008 (WO11/140249; WO13169264; WO14/036357).

Anti-TIM3 antibodies can also be administered with agents that inhibitTGF-β signaling.

Additional agents that can be combined with an anti-TIM3 antibodyinclude agents that enhance tumor antigen presentation, e.g., dendriticcell vaccines, GM-CSF secreting cellular vaccines, CpG oligonucleotides,and imiquimod, or therapies that enhance the immunogenicity of tumorcells (e.g., anthracyclines).

Yet other therapies that can be combined with an anti-TIM3 antibodyinclude therapies that deplete or block Treg cells, e.g., an agent thatspecifically binds to CD25.

Another therapy that can be combined with an anti-TIM3 antibody is atherapy that inhibits a metabolic enzyme such as indoleamine dioxigenase(IDO), dioxigenase, arginase, or nitric oxide synthetase.

Another class of agents that can be used with an anti-TIM3 antibodyincludes agents that inhibit the formation of adenosine, e.g., CD73inhibitors, or inhibit the adenosine A2A receptor.

Other therapies that can be combined with an anti-TIM3 antibody fortreating cancer include therapies that reverse/prevent T cell anergy orexhaustion and therapies that trigger an innate immune activation and/orinflammation at a tumor site.

Other therapies that can be combined with an anti-TIM3 antibody fortreating cancer include therapies that block IL-8, e.g., with HuMax-IL8.

An anti-TIM3 antibody can be combined with more than one immuno-oncologyagent, and can be, e.g., combined with a combinatorial approach thattargets multiple elements of the immune pathway, such as one or more ofthe following: a therapy that enhances tumor antigen presentation (e.g.,dendritic cell vaccine, GM-CSF secreting cellular vaccines, CpGoligonucleotides, imiquimod); a therapy that inhibits negative immuneregulation e.g., by inhibiting CTLA-4 and/or PD 1/PD-L1/PD-L2 pathwayand/or depleting or blocking Tregs or other immune suppressing cells; atherapy that stimulates positive immune regulation, e.g., with agoniststhat stimulate the CD-137, OX-40, and/or CD40 or GITR pathway and/orstimulate T cell effector function; a therapy that increasessystemically the frequency of anti-tumor T cells; a therapy thatdepletes or inhibits Tregs, such as Tregs in the tumor, e.g., using anantagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 beaddepletion; a therapy that impacts the function of suppressor myeloidcells in the tumor; a therapy that enhances immunogenicity of tumorcells (e.g., anthracyclines); adoptive T cell or NK cell transferincluding genetically modified cells, e.g., cells modified by chimericantigen receptors (CAR-T therapy); a therapy that inhibits a metabolicenzyme such as indoleamine dioxigenase (IDO), dioxigenase, arginase, ornitric oxide synthetase; a therapy that reverses/prevents T cell anergyor exhaustion; a therapy that triggers an innate immune activationand/or inflammation at a tumor site; administration of immunestimulatory cytokines; or blocking of immuno repressive cytokines.

Anti-TIM3 antibodies described herein can be used together with one ormore of agonistic agents that ligate positive costimulatory receptors,blocking agents that attenuate signaling through inhibitory receptors,antagonists, and one or more agents that increase systemically thefrequency of anti-tumor T cells, agents that overcome distinct immunesuppressive pathways within the tumor microenvironment (e.g., blockinhibitory receptor engagement (e.g., PD-L1/PD-1 interactions), depleteor inhibit Tregs (e.g., using an anti-CD25 monoclonal antibody (e.g.,daclizumab) or by ex vivo anti-CD25 bead depletion), inhibit metabolicenzymes such as IDO, or reverse/prevent T cell anergy or exhaustion) andagents that trigger innate immune activation and/or inflammation attumor sites.

In certain embodiments, an anti-TIM3 antibody is administered to asubject together with a BRAF inhibitor if the subject is BRAF V600mutation positive.

Suitable PD-1 antagonists for use in the combination therapy describedherein, include, without limitation, ligands, antibodies (e.g.,monoclonal antibodies and bispecific antibodies), and multivalentagents. In one embodiment, the PD-1 antagonist is a fusion protein,e.g., an Fc fusion protein, such as AMP-244. In one embodiment, the PD-1antagonist is an anti-PD-1 or anti-PD-L1 antibody.

An exemplary anti-PD-1 antibody is nivolumab (BMS-936558) or an antibodythat comprises the CDRs or variable regions of one of antibodies 17D8,2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 described in WO 2006/121168. In certainembodiments, an anti-PD-1 antibody is MK-3475 (Lambrolizumab) describedin WO2012/145493; AMP-514 described in WO 2012/145493; or PDR001.Further known PD-1 antibodies and other PD-1 inhibitors include thosedescribed in WO 2009/014708, WO 03/099196, WO 2009/114335, WO2011/066389, WO 2011/161699, WO 2012/145493, U.S. Pat. Nos. 7,635,757and 8,217,149, and U.S. Patent Publication No. 2009/0317368. Any of theanti-PD-1 antibodies disclosed in WO2013/173223 can also be used. Ananti-PD-1 antibody that competes for binding with, and/or binds to thesame epitope on PD-1 as, as one of these antibodies can also be used incombination treatments.

In some embodiments, the anti-PD-L1 antibody useful for the combinationtherapy is BMS-936559 (referred to as 12A4 in WO 2007/005874 and U.S.Pat. No. 7,943,743), or an antibody that comprises the CDRs or variableregions of 3G10, 12A4, 10A5, 5F8, 10H10, 1B 12, 7H1, 11E6, 12B7 and13G4, which are described in PCT Publication WO 07/005874 and U.S. Pat.No. 7,943,743. In certain embodiment an anti-PD-L1 antibody is MEDI4736(also known as durvalumab and Anti-B7-H1), MPDL3280A (also known asatezolizumab and RG7446), MSB0010718C (also known as avelumab;WO2013/79174), or rHigM12B7. Any of the anti-PD-L1 antibodies disclosedin WO2013/173223, WO2011/066389, WO2012/145493, U.S. Pat. Nos. 7,635,757and 8,217,149 and U.S. Publication No. 2009/145493 can also be used.Anti-PD-L1 antibodies that compete with and/or bind to the same epitopeas that of any of these antibodies can also be used in combinationtreatments.

In certain embodiments, the anti-TIM3 antibody of the disclosure can beused with a CTLA-4 antagonist, e.g., an anti-CTLA-4 antibody. In oneembodiment, an anti-CTLA-4 antibody is an antibody selected from thegroup of: YERVOY® (ipilimumab or antibody 10D1, described in PCTPublication WO 01/14424), tremelimumab (formerly ticilimumab,CP-675,206), monoclonal or an anti-CTLA-4 antibody described in any ofthe following publications: WO 98/42752; WO 00/37504; U.S. Pat. No.6,207,156; Hurwitz et al. (1998) Pro. Natl. Acad. Sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): AbstractNo. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res.58:5301-5304. Any of the anti-CTLA-4 antibodies disclosed inWO2013/173223 can also be used.

In other embodiments, an anti-TIM3 antibody of the disclosure is used incombination with a LAG3 antagonist. Examples of anti-LAG3 antibodiesinclude antibodies comprising the CDRs or variable regions of antibodies25F7, 26H10, 25E3, 8B7, 11F2 or 17E5, which are described in U.S. PatentPublication No. US2011/0150892, WO10/19570 and WO2014/008218. In oneembodiment, an anti-LAG-3 antibody is BMS-986016. Other art recognizedanti-LAG-3 antibodies that can be used include IMP731 and IMP-321,described in US 2011/007023, WO08/132601, and WO09/44273. Anti-LAG-3antibodies that compete with and/or bind to the same epitope as that ofany of these antibodies can also be used in combination treatments.

In some embodiments, an anti-TIM3 antibody of the disclosure can beadministered in combination with a CD137 (4-1BB) agonist, such as anagonistic CD137 antibody. Suitable CD137 antibodies include, forexample, urelumab or PF-05082566 (WO12/32433).

In other embodiments, an anti-TIM3 antibody can be administered incombination with an OX40 agonist, such as an agonistic OX40 antibody.Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469 orMOXR0916 (RG7888; WO06/029879).

In one embodiment, an anti-TIM3 antibody is administered in combinationwith a CD40 agonist, such as an agonistic CD40 antibody. In certainembodiments, the immuno-oncology agent is a CD40 antagonist, such as anantagonistic CD40 antibody. Suitable CD40 antibodies include, forexample, lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893 or ChiLob 7/4.

In one embodiment, an anti-TIM3 antibody is administered in combinationwith a CD27 agonist, such as an agonistic CD27 antibody. Suitable CD27antibodies include, for example, varlilumab (CDX-1127).

In certain embodiments, the anti-TIM3 antibody is administered togetherwith an anti-GITR antibody, e.g., an antibody having the CDR sequencesof 6C8, e.g., a humanized antibody having the CDRs of 6C8, as described,e.g., in WO2006/105021; an antibody comprising the CDRs of an anti-GITRantibody described in WO2011/028683; an antibody comprising the CDRs ofan anti-GITR antibody described in JP2008278814, an antibody comprisingthe CDRs of an anti-GITR antibody described in WO2015/031667,WO2015/187835, WO2015/184099, WO2016/054638, WO2016/057841 orWO2016/057846 or other anti-GITR antibody described or referred toherein.

In other embodiments, an anti-TIM3 antibody is administered incombination with MGA271 (to B7H3) (WO11/109400).

In some embodiments, an anti-TIM3 antibody is administered incombination with a KIR antagonist, such as lirilumab.

In other embodiments, an anti-TIM3 antibody is administered incombination with an IDO antagonist. Suitable IDO antagonists include,for example, INCB-024360 (WO2006/122150, WO07/75598, WO08/36653,WO08/36642), indoximod, NLG-919 (WO09/73620, WO09/1156652, WO11/56652,WO12/142237) or F001287.

In yet other embodiments, an anti-TIM3 antibody is administered incombination with a Toll-like receptor agonist, e.g., a TLR2/4 agonist(e.g., Bacillus Calmette-Guerin); a TLR7 agonist (e.g., Hiltonol orImiquimod); a TLR7/8 agonist (e.g., Resiquimod); or a TLR9 agonist(e.g., CpG7909).

In one embodiment, an anti-TIM3 is administered in combination with aTGF-β inhibitor, e.g., GC1008, LY2157299, TEW7197, or IMC-TR1.

The anti-TIM3 antibodies and combination therapies described herein canalso be used in conjunction with other well-known therapies that areselected for their particular usefulness against the indication beingtreated (e.g., cancer). Combinations of the anti-TIM3 antibodiesdescribed herein can be used sequentially with known pharmaceuticallyacceptable agent(s).

For example, the anti-TIM3 antibodies and combination therapiesdescribed herein can be used in combination (e.g., simultaneously orseparately) with an additional treatment, such as irradiation and/orchemotherapy, e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU),cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin,paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, orcamptothecin+apo21/TRAIL (a 6× combo)), one or more proteasomeinhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors(e.g., BH3I-2′ (bcl-x1 inhibitor), indoleamine dioxygenase-1 inhibitor(e.g., INCB24360, indoximod, NLG-919, or F001287), AT-101(R-(−)-gossypol derivative), ABT-263 (small molecule), GX-15-070(obatoclax), or MCL-1 (myeloid leukemia cell differentiation protein-1)antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g.,smac7, smac4, small molecule smac mimetic, synthetic smac peptides (seeFulda et al., Nat Med 2002; 8:808-15), ISIS23722 (LY2181308), orAEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20antibodies (e.g., rituximab), angiogenesis inhibitors (e.g.,bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g.,Avastin), synthetic triterpenoids (see Hyer et al, Cancer Research 2005;65:4799-808), c-FLIP (cellular FLICE-inhibitory protein) modulators(e.g., natural and synthetic ligands of PPARy (peroxisomeproliferator-activated receptor γ), 5809354 or 5569100), kinaseinhibitors (e.g., Sorafenib), Trastuzumab, Cetuximab, Temsirolimus, mTORinhibitors such as rapamycin and temsirolimus, Bortezomib, JAK2inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, Lenalildomide, GSK3Pinhibitors, IAP inhibitors and/or genotoxic drugs.

The anti-TIM3 antibodies and combination therapies described herein canfurther be used in combination with one or more anti-proliferativecytotoxic agents. Classes of compounds that can be used asanti-proliferative cytotoxic agents include, but are not limited to, thefollowing:

Alkylating agents (including, without limitation, nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes):Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN®) fosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists,pyrimidine analogs, purine analogs and adenosine deaminase inhibitors):Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for combining with anti-TIM3antibodies, without limitation, taxanes, paclitaxel (paclitaxel iscommercially available as TAXOL™), docetaxel, discodermolide (DDM),dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothiloneB, epothilone C, epothilone D, epothilone E, epothilone F,furanoepothilone D, desoxyepothilone B1, [17]-dehydrodesoxyepothilone B,[18]dehydrodesoxyepothilones B, C12,13-cyclopropyl-epothilone A, C6-C8bridged epothilone A, trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone BIO,discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO,ABJ-789, XAA296A (Discodermolide), TZT-1027 (soblidotin), ILX-651(tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389),Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoidimmunoconjugates (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992(ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin,17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,cyclostreptin, isolaulimalide, laulimalide,4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, andcryptothilone 1, in addition to other microtubuline stabilizing agentsknown in the art.

In cases where it is desirable to render aberrantly proliferative cellsquiescent in conjunction with or prior to treatment with anti-TIM3antibodies described herein, hormones and steroids (including syntheticanalogs), such as 17a-Ethinylestradiol, Diethylstilbestrol,Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate,Testolactone, Megestrolacetate, Methylprednisolone, Methyl-testosterone,Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone,Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide,Flutamide, Toremifene, ZOLADEX®, can also be administered to thepatient. When employing the methods or compositions described herein,other agents used in the modulation of tumor growth or metastasis in aclinical setting, such as antimimetics, can also be administered asdesired.

In certain embodiments, the combination of the anti-TIM3 antibody and asecond agent discussed herein can be administered concurrently as asingle composition in a pharmaceutically acceptable carrier, orconcurrently as separate compositions with the anti-TIM3 antibody andthe second agent in a pharmaceutically acceptable carrier. In anotherembodiment, the combination of the anti-TIM3 antibody and the secondagent can be administered sequentially. The administration of the twoagents can start at times that are, e.g., 30 minutes, 60 minutes, 90minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48hours, 3 days, 5 days, 7 days, or one or more weeks apart, oradministration of the second agent can start, e.g., 30 minutes, 60minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours,36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks afterthe first agent has been administered.

In some embodiments, an anti-neoplastic antibody that can be combinedwith an anti-TIM3 antibody and/or a second agent includes RITUXAN®(rituximab), HERCEPTIN® (trastuzumab), BEXXAR® (tositumomab), ZEVALIN®(ibritumomab), CAMPATH® (alemtuzumab), LYMPHOCIDE® (eprtuzumab),AVASTIN® (bevacizumab), and TARCEVA® (erlotinib), or any combinationthereof. In other embodiments, the second antibody useful for thecombination therapy with an anti-TIM3 antibody can be an antibody drugconjugate.

In other embodiment, an anti-TIM3 antibody alone or in combination withanother agent is used concurrently or sequentially with bone marrowtransplantation to treat a variety of tumors of hematopoietic origin.

Provided herein are methods for altering an adverse event associatedwith treatment of a hyperproliferative disease (e.g., cancer) with animmuno stimulatory agent, comprising administering an anti-TIM3 antibodywith or without a second agent, to a subject. For example, the methodsdescribed herein provide for a method of reducing the incidence ofimmuno stimulatory therapeutic antibody-induced colitis or diarrhea byadministering a non-absorbable steroid to the patient. As used herein, a“non-absorbable steroid” is a glucocorticoid that exhibits extensivefirst pass metabolism such that, following metabolism in the liver, thebioavailability of the steroid is low, i.e., less than about 20%. In oneembodiment described herein, the non-absorbable steroid is budesonide.Budesonide is a locally-acting glucocorticosteroid, which is extensivelymetabolized, primarily by the liver, following oral administration.ENTOCORT EC® (Astra-Zeneca) is a pH- and time-dependent oral formulationof budesonide developed to optimize drug delivery to the ileum andthroughout the colon. ENTOCORT EC® is approved in the U.S. for thetreatment of mild to moderate Crohn's disease involving the ileum and/orascending colon. In still further embodiments, an anti-TIM3 antibody inconjunction with a non-absorbable steroid can be further combined with asalicylate. Salicylates include 5-ASA agents such as, for example:sulfasalazine (AZULFIDINE®, Pharmacia & Up John); olsalazine (DJPENTUM®,Pharmacia & Up John); balsalazide (COLAZAL®, Salix Pharmaceuticals,Inc.); and mesalamine (ASACOL®, Procter & Gamble Pharmaceuticals;PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay).

TABLE 1 SEQ ID Description Sequences 1 TIM3.5 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.1f HeavyKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPS ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 28B9 IgG1.1fQVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIGYIHYSGSTNYNSSLKSHeavy ChainRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDTGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 3TIM3.6 (8C4)QVQLQESGPGLVKPSETLSLTCTVSGGSISRYYWSWIRQPPGKGLEWIGYIHYTGSTNYNPSLKSIgG1.1f HeavyRVTISVDTSKNQFSLKLSSVTAADTAVYYCATDTGYYGMDVWGQGTTVTVSSASTKGPSVFPLAP ChainSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 4TIM3.2 (17C3)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPRGDSIIYAQKFQIgG1.1f HeavyGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDFYGSGNYYYGMDVWGQGTTVTVSSASTKGPS ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 59F6 IgG1.1fQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISGGGSTIYYADSVKHeavy ChainGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCARDGYSSGWYYYGMDVWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 6TIM3.4 (3G4)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISTSGSIIYYADSVKIgG1.1f HeavyGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGYSSSWSYYYGMDVWGQGTTVTVSSASTKGP ChainSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 7TIM3.9 (17C8)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISSSGSIIYYADSVKIgG1.1f HeavyGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGYSSGWEYYGMDVWGQGTTVTVSSASTKGPS ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 8TIM3.5 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.1f HeavyKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSChain (withoutVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 98B9 IgG1.1fQVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIGYIHYSGSTNYNSSLKSHeavy ChainRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDTGYYGMDIWGQGTTVTVSSASTKGPSVFPLAP(without C-SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTterminal K)QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 10TIM3.6 (8C4)QVQLQESGPGLVKPSETLSLTCTVSGGSISRYYWSWIRQPPGKGLEWIGYIHYTGSTNYNPSLKSIgG1.1f HeavyRVTISVDTSKNQFSLKLSSVTAADTAVYYCATDTGYYGMDVWGQGTTVTVSSASTKGPSVFPLAPChain (withoutSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTC-terminal K)QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 11TIM3.2 (17C3)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPRGDSIIYAQKFQIgG1.1f HeavyGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDFYGSGNYYYGMDVWGQGTTVTVSSASTKGPSChain (withoutVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 129F6 IgG1.1fQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISGGGSTIYYADSVKHeavy ChainGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCARDGYSSGWYYYGMDVWGQGTAVTVSSASTKGPS(without C-VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPterminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 13TIM3.4 (3G4)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISTSGSIIYYADSVKIgG1.1f HeavyGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGYSSSWSYYYGMDVWGQGTTVTVSSASTKGPChain (withoutSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVC-terminal K)PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 14TIM3.9 (17C8)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISSSGSIIYYADSVKIgG1.1f HeavyGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGYSSGWEYYGMDVWGQGTTVTVSSASTKGPSChain (withoutVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 15TIM3.5 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.3f HeavyKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPS ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 168B9 IgG1.3fQVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIGYIHYSGSTNYNSSLKSHeavy ChainRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDTGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 17TIM3.6 (8C4)QVQLQESGPGLVKPSETLSLTCTVSGGSISRYYWSWIRQPPGKGLEWIGYIHYTGSTNYNPSLKSIgG1.3f HeavyRVTISVDTSKNQFSLKLSSVTAADTAVYYCATDTGYYGMDVWGQGTTVTVSSASTKGPSVFPLAP ChainSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 18TIM3.2 (17C3)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPRGDSIIYAQKFQIgG1.3f HeavyGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDFYGSGNYYYGMDVWGQGTTVTVSSASTKGPS ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 199F6 IgG1.3fQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISGGGSTIYYADSVKHeavy ChainGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCARDGYSSGWYYYGMDVWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 20TIM3.4 (3G4)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISTSGSIIYYADSVKIgG1.3f HeavyGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGYSSSWSYYYGMDVWGQGTTVTVSSASTKGP ChainSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 21TIM3.9 (17C8)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISSSGSIIYYADSVKIgG1.3f HeavyGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGYSSGWEYYGMDVWGQGTTVTVSSASTKGPS ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 22TIM3.5 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.3f HeavyKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSChain (no C-VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPterminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 238B9 IgG1.3fQVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIGYIHYSGSTNYNSSLKSHeavy Chain (noRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDTGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPC-terminal K)SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 24TIM3.6 (8C4)QVQLQESGPGLVKPSETLSLTCTVSGGSISRYYWSWIRQPPGKGLEWIGYIHYTGSTNYNPSLKSIgG1.3f HeavyRVTISVDTSKNQFSLKLSSVTAADTAVYYCATDTGYYGMDVWGQGTTVTVSSASTKGPSVFPLAPChain (no C-SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTterminal K)QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 25TIM3.2 (17C3)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPRGDSIIYAQKFQIgG1.3f HeavyGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDFYGSGNYYYGMDVWGQGTTVTVSSASTKGPSChain (no C-VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPterminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 269F6 IgG1.3fQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISGGGSTIYYADSVKHeavy Chain (noGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCARDGYSSGWYYYGMDVWGQGTAVTVSSASTKGPSC-terminal K)VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 27TIM3.4 (3G4)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISTSGSIIYYADSVKIgG1.3f HeavyGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGYSSSWSYYYGMDVWGQGTTVTVSSASTKGPChain (no C-SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVterminal K)PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 28TIM3.9 (17C8)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISSSGSIIYYADSVKIgG1.3f HeavyGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGYSSGWEYYGMDVWGQGTTVTVSSASTKGPSChain (no C-VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPterminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 72TIM3.10 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.1f (N60Q)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 73TIM3.11 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYSPSLIgG1.1f (N60S)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 74TIM3.12 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYAPSLIgG1.1f (N60A)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 75TIM3.13 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.1f (D101E)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 76TIM3.14 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.1f (P102V)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDVWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 77TIM3.15 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.1f (P102Y)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 78TIM3.16 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.1f (P102L)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDLWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 79TIM3.17 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.1fKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDYWGQGTLVTVSSASTKGPS(N60Q/P102Y)VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPHeavy ChainSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 349TIM3.18 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.1fKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKGPS(N60Q/D101E)VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPHeavy ChainSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 80TIM3.8 (8B9)QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIGYIHYSGSTNYNPSLKSIgG1.1f (S61P)RVTISVDTSKNQFSLKLSSVTAADTAVYYCARDTGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPHeavy ChainSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 81TIM3.7 (9F6)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISGGGSTIYYADSVKIgG1.1f (A108T)GRFTISRDNAKNSLFLQMNSLRVEDTAVYYCARDGYSSGWYYYGMDVWGQGTTVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 82TIM3.10 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.1f (N60Q)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 83TIM3.11 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYSPSLIgG1.1f (N60S)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 84TIM3.12 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYAPSLIgG1.1f (N60A)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 85TIM3.13 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.1f (D101E)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 86TIM3.14 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.1f (P102V)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDVWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 87TIM3.15 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.1f (P102Y)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 88TIM3.16 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.1f (P102L)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDLWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 89TIM3.17 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.1fKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDYWGQGTLVTVSSASTKGPS(N60Q/P102Y)VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPHeavy Chain (noSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISC-terminal K)RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 350TIM3.18 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.1fKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKGPS(N60Q/D101E)VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPHeavy Chain (noSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISC-terminal K)RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 90TIM3.8 (8B9)QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIGYIHYSGSTNYNPSLKSIgG1.1f (S61P)RVTISVDTSKNQFSLKLSSVTAADTAVYYCARDTGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPHeavy Chain (noSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTC-terminal K)QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 91TIM3.7 (9F6)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISGGGSTIYYADSVKIgG1.1f (A108T)GRFTISRDNAKNSLFLQMNSLRVEDTAVYYCARDGYSSGWYYYGMDVWGQGTTVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 92TIM3.10 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.3f (N60Q)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 93TIM3.11 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYSPSLIgG1.3f (N60S)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 94TIM3.12 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYAPSLIgG1.3f (N60A)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 95TIM3.13 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.3f (D101E)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 96TIM3.14 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.3f (P102V)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDVWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 97TIM3.15 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.3f (P102Y)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 98TIM3.16 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.3f (P102L)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDLWGQGTLVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 99TIM3.17 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.3fKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDYWGQGTLVTVSSASTKGPS(N60Q/P102Y)VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPHeavy ChainSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 351TIM3.18 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.3fKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKGPS(N60Q/D101E)VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPHeavy ChainSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 100TIM3.8 (8B9)QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIGYIHYSGSTNYNPSLKSIgG1.3f (S61P)RVTISVDTSKNQFSLKLSSVTAADTAVYYCARDTGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPHeavy ChainSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 101TIM3.7 (9F6)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISGGGSTIYYADSVKIgG1.3f (A108T)GRFTISRDNAKNSLFLQMNSLRVEDTAVYYCARDGYSSGWYYYGMDVWGQGTTVTVSSASTKGPSHeavy ChainVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 102TIM3.10 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.3f (N60Q)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 103TIM3.11 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYSPSLIgG1.3f (N60S)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 104TIM3.12 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYAPSLIgG1.3f (N60A)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 105TIM3.13 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.3f (D101E)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 106TIM3.14 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.3f (P102V)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDVWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 107TIM3.15 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.3f (P102Y)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 108TIM3.16 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLIgG1.3f (P102L)KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDLWGQGTLVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 109TIM3.17 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.3fKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDYWGQGTLVTVSSASTKGPS(N60Q/P102Y)VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPHeavy Chain (noSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISC-terminal K)RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 352TIM3.18 (13A3)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLIgG1.3fKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKGPS(N60Q/D101E)VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPHeavy Chain (noSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISC-terminal K)RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 110TIM3.8 (8B9)QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIGYIHYSGSTNYNPSLKSIgG1.3f (S61P)RVTISVDTSKNQFSLKLSSVTAADTAVYYCARDTGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPHeavy Chain (noSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTC-terminal K)QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 111TIM3.7 (9F6)QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSFISGGGSTIYYADSVKIgG1.3f (A108T)GRFTISRDNAKNSLFLQMNSLRVEDTAVYYCARDGYSSGWYYYGMDVWGQGTTVTVSSASTKGPSHeavy Chain (noVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPC-terminal K)SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* 29TIM3.5 (13A3),EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGTIM3.2 (17C3),SGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTTIM3.4 (3G4)ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACIgG1 Light EVTHQGLSSPVTKSFNRGEC* Chain 30 8B9, TIM3.6EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSG(8C4), TIM3.9SGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT(17C8) IgG1ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACLight Chain EVTHQGLSSPVTKSFNRGEC* 32 9F6 VK1 IgG1AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSLight ChainGSGTDFTLTISSLQPEDFATYYCQQFNSYPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* 134 TIM3.5 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f HeavyCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAG ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 1358B9 IgG1.1fCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGHeavy ChainCACTGTCTCTGGTGGCTCCATCAGTCGTCACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCCATTACAGTGGAAGCACCAACTACAATTCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 136 TIM3.6 (8C4)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f HeavyCACTGTCTCTGGTGGCTCCATCAGTCGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGG ChainGACTGGAGTGGATTGGGTATATCCATTACACTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCAGCGGACACGGCCGTGTATTACTGTGCGACAGATACGGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 137 TIM3.2 (17C3)CAGGTGCAGTTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGIgG1.1f HeavyCAAGGCATCTGGATACACTTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAG ChainGGCTTGAGTGGATGGGAATAATCAACCCTAGGGGTGATAGCATAATCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTCTATGGTTCGGGAAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 1389F6 IgG1.1fCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGHeavy ChainTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCTGTTTCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCGCGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 139TIM3.4 (3G4)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.1f HeavyTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGG ChainGGCTGGAGTGGGTTTCATTCATTAGTACTAGTGGTAGTATCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAAGGGTATAGCAGCAGCTGGTCCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 140TIM3.9 (17C8)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.1f HeavyTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGG ChainGGCTGGAGTGGGTTTCATTCATTAGTAGTAGTGGTAGTATCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGTATAGCAGTGGCTGGGAGTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 141TIM3.5 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f HeavyCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGChain (withoutGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 1428B9 IgG1.1fCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGHeavy ChainCACTGTCTCTGGTGGCTCCATCAGTCGTCACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGG(without C-GACTGGAGTGGATTGGGTATATCCATTACAGTGGAAGCACCAACTACAATTCCTCCCTCAAGAGTterminal K)CGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 143 TIM3.6 (8C4)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f HeavyCACTGTCTCTGGTGGCTCCATCAGTCGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGChain (withoutGACTGGAGTGGATTGGGTATATCCATTACACTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTC-terminal K)CGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCAGCGGACACGGCCGTGTATTACTGTGCGACAGATACGGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 144 TIM3.2 (17C3)CAGGTGCAGTTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGIgG1.1f HeavyCAAGGCATCTGGATACACTTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGChain (withoutGGCTTGAGTGGATGGGAATAATCAACCCTAGGGGTGATAGCATAATCTACGCACAGAAGTTCCAGC-terminal K)GGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTCTATGGTTCGGGAAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 1459F6 IgG1.1fCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGHeavy ChainTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGG(without C-GGCTGGAGTGGGTTTCATTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGterminal K)GGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCTGTTTCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCGCGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 146TIM3.4 (3G4)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.1f HeavyTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGChain (withoutGGCTGGAGTGGGTTTCATTCATTAGTACTAGTGGTAGTATCATATACTACGCAGACTCTGTGAAGC-terminal K)GGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAAGGGTATAGCAGCAGCTGGTCCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 147TIM3.9 (17C8)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.1f HeavyTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGChain (withoutGGCTGGAGTGGGTTTCATTCATTAGTAGTAGTGGTAGTATCATATACTACGCAGACTCTGTGAAGC-terminal K)GGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGTATAGCAGTGGCTGGGAGTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 148TIM3.5 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f HeavyCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAG ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 1498B9 IgG1.3fAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCHeavy ChainACTGTCTCTGGTGGCTCCATCAGTCGTCACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCCATTACAGTGGAAGCACCAACTACAATTCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 150 TIM3.6 (8C4)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f HeavyCACTGTCTCTGGTGGCTCCATCAGTCGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGG ChainGACTGGAGTGGATTGGGTATATCCATTACACTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCAGCGGACACGGCCGTGTATTACTGTGCGACAGATACGGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 151 TIM3.2 (17C3)CAGGTGCAGTTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGIgG1.3f HeavyCAAGGCATCTGGATACACTTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAG ChainGGCTTGAGTGGATGGGAATAATCAACCCTAGGGGTGATAGCATAATCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTCTATGGTTCGGGAAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 1529F6 IgG1.3fCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGHeavy ChainTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCTGTTTCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCGCGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 153TIM3.4 (3G4)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.3f HeavyTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGG ChainGGCTGGAGTGGGTTTCATTCATTAGTACTAGTGGTAGTATCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAAGGGTATAGCAGCAGCTGGTCCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 154TIM3.9 (17C8)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.3f HeavyTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGG ChainGGCTGGAGTGGGTTTCATTCATTAGTAGTAGTGGTAGTATCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGTATAGCAGTGGCTGGGAGTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 155TIM3.5 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f HeavyCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGChain (no C-GGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCterminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 1568B9 IgG1.3fAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCHeavy Chain (noACTGTCTCTGGTGGCTCCATCAGTCGTCACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGC-terminal K)ACTGGAGTGGATTGGGTATATCCATTACAGTGGAAGCACCAACTACAATTCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 157 TIM3.6 (8C4)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f HeavyCACTGTCTCTGGTGGCTCCATCAGTCGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGChain (no C-GACTGGAGTGGATTGGGTATATCCATTACACTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTterminal K)CGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCAGCGGACACGGCCGTGTATTACTGTGCGACAGATACGGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 158 TIM3.2 (17C3)CAGGTGCAGTTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGIgG1.3f HeavyCAAGGCATCTGGATACACTTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGChain (no C-GGCTTGAGTGGATGGGAATAATCAACCCTAGGGGTGATAGCATAATCTACGCACAGAAGTTCCAGterminal K)GGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTCTATGGTTCGGGAAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 1599F6 IgG1.3fCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGHeavy Chain (noTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGC-terminal K)GGCTGGAGTGGGTTTCATTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCTGTTTCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCGCGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 160TIM3.4 (3G4)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.3f HeavyTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGChain (no C-GGCTGGAGTGGGTTTCATTCATTAGTACTAGTGGTAGTATCATATACTACGCAGACTCTGTGAAGterminal K)GGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAAGGGTATAGCAGCAGCTGGTCCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 161TIM3.9 (17C8)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.3f HeavyTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGChain (no C-GGCTGGAGTGGGTTTCATTCATTAGTAGTAGTGGTAGTATCATATACTACGCAGACTCTGTGAAGterminal K)GGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGTATAGCAGTGGCTGGGAGTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 205TIM3.10 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (N60Q)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 206TIM3.11 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (N60S)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACTCACCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 207TIM3.12 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (N60A)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACGCACCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 208TIM3.13 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (D101E)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 209TIM3.14 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (P102V)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACGTATGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 210TIM3.15 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (P102Y)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 211TIM3.16 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (P102L)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCTATGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 212TIM3.17 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1fCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAG(N60Q/P102Y)GGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCHeavy ChainAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 355TIM3.18 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1fCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAG(N60Q/D101E)GGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCHeavy ChainAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 213TIM3.8 (8B9)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (S61P)CACTGTCTCTGGTGGCTCCATCAGTCGTCACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGHeavy ChainGACTGGAGTGGATTGGGTATATCCATTACAGTGGAAGCACCAACTACAATCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 214 TIM3.7 (9F6)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.1f (A108T)TGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGHeavy ChainGGCTGGAGTGGGTTTCATTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCTGTTTCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 215TIM3.10 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (N60Q)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 216TIM3.11 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (N60S)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACTCACCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 217TIM3.12 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (N60A)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACGCACCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 218TIM3.13 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (D101E)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 219TIM3.14 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (P102V)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACGTATGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 220TIM3.15 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (P102Y)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 221TIM3.16 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (P102L)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCTATGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 222TIM3.17 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1fCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAG(N60Q/P102Y)GGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCHeavy Chain (noAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTC-terminal K)GACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 356TIM3.18 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1fCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAG(N60Q/D101E)GGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCHeavy Chain (noAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTC-terminal K)GACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 223TIM3.8 (8B9)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.1f (S61P)CACTGTCTCTGGTGGCTCCATCAGTCGTCACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGHeavy Chain (noGACTGGAGTGGATTGGGTATATCCATTACAGTGGAAGCACCAACTACAATCCCTCCCTCAAGAGTC-terminal K)CGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 224 TIM3.7 (9F6)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.1f (A108T)TGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGHeavy Chain (noGGCTGGAGTGGGTTTCATTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGC-terminal K)GGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCTGTTTCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 225TIM3.10 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (N60Q)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 226TIM3.11 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (N60S)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACTCACCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 227TIM3.12 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (N60A)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACGCACCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 228TIM3.13 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (D101E)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 229TIM3.14 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (P102V)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACGTATGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 230TIM3.15 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (P102Y)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 231TIM3.16 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (P102L)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy ChainGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCTATGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 232TIM3.17 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3fCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAG(N60Q/P102Y)GGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCHeavy ChainAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 357TIM3.18 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3fCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAG(N60Q/D101E)GGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCHeavy ChainAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 233TIM3.8 (8B9)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (S61P)CACTGTCTCTGGTGGCTCCATCAGTCGTCACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGHeavy ChainGACTGGAGTGGATTGGGTATATCCATTACAGTGGAAGCACCAACTACAATCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 234 TIM3.7 (9F6)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.3f (A108T)TGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGHeavy ChainGGCTGGAGTGGGTTTCATTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCTGTTTCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 235TIM3.10 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (N60Q)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 236TIM3.11 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (N60S)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACTCACCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 237TIM3.12 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (N60A)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACGCACCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 238TIM3.13 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (D101E)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 239TIM3.14 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (P102V)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACGTATGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 240TIM3.15 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (P102Y)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 241TIM3.16 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (P102L)CACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGHeavy Chain (noGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCC-terminal K)AAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCTATGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 242TIM3.17 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3fCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAG(N60Q/P102Y)GGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCHeavy Chain (noAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTC-terminal K)GACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 358TIM3.18 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3fCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAG(N60Q/D101E)GGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCHeavy Chain (noAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTC-terminal K)GACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 374TIM3.18 (13A3)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3fCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAGTTACTACTGGGGCTGGATTCGCCAGCCCCCAG(N60Q/D101E)GGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTC(T168C) HeavyAAGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTChain (no C-GACCGCCGCAGACACGGCTGTGTATTATTGTGCGACAGGGGGGCCCTACGGTGACTACGCCCACTterminal K)GGTTCGAACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 243TIM3.8 (8B9)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGIgG1.3f (S61P)CACTGTCTCTGGTGGCTCCATCAGTCGTCACTACTGGAACTGGATCCGGCAGCCCCCAGGGAAGGHeavy Chain (noGACTGGAGTGGATTGGGTATATCCATTACAGTGGAAGCACCAACTACAATCCCTCCCTCAAGAGTC-terminal K)CGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 244 TIM3.7 (9F6)CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGIgG1.3f (A108T)TGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGHeavy Chain (noGGCTGGAGTGGGTTTCATTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGC-terminal K)GGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCTGTTTCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 162TIM3.5 (13A3),GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCTIM3.2 (17C3),CTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGTIM3.4 (3G4)CTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCIgG1 LightAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA ChainTTACTGTCAGCAGTATGGTAGCTCACCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG 1638B9, TIM3.6GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTC(8C4), TIM3.9CTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGG(17C8) IgG1CTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCLight ChainAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG 1659F6 VK1 IgG1GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACLight ChainTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG 1669F6 VK2 IgG1GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCLight ChainCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG 1649F6 VK3 IgG1GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCLight ChainCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); ); Crooks, Antisense drug Technology:Principles, strategies and applications, 2^(nd) Ed. CRC Press (2007) andin Ausubel et al. (1989) Current Protocols in Molecular Biology (JohnWiley and Sons, Baltimore, Md.).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1: Identification of Human Anti-TIM3 Antibodies

Human IgG transgenic (KM) mice were immunized with the plasma membranefraction of HEK-293 human cells transfected with human TIM-3. Lymph nodecells from all immunized mice were fused to the SP2/0 fusion partner.Hybridoma supernatants were first screened for the presence of human IgGantibodies using a high throughput assay. Antigen specificity was thendetermined by FACS binding on human TIM-3 transfected cells. Briefly, 47fusions were performed, 3935 IgG positive clones were identified, ofwhich 448 were identified as being positive for hTIM3 by ELISA, and ofthese 126 were found to be positive by hTIM3 FACS. Of these, 117 clones(or antibodies) were further analyzed by a variety of methods including:(1) epitope binning performed by Biacore; (2) TIM3 binding to aTIM-3-transfected cell line (293-TIM3) to determine EC₅₀; (3) Th1 assays(as further described below); and (4) TIL assays (as further describedbelow). Of the 117, seven hybridomas expressing fully human anti-humanTIM3 antibodies were selected as having desirable characteristics: 13A3,8B9, 8C4, 17C3, 9F6, 3G4 and 17C8. The amino acid of, and nucleotidesequences encoding, the variable domains of the antibodies produced bythese hybridomas are provided in FIGS. 1-7, and the SEQ ID NOs of theCDRs, variable regions and heavy and light chains as well as theirisotype are provided in FIG. 13 (see rows with hybridoma names). Ahydriboma and antibody secreted by it have the same name (e.g., 13A3).

Antibodies comprising the CDRs and/or variable domains of antibodies13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 were also expressedrecombinantly in host cells. Recombinant antibodies are referred toherein with the names “TIM3.2” to “TIM3.18.” When referring to any ofthese recombinant antibodies by their names “TIM3.2” to “TIM3.18”, nospecific constant region is referred to, i.e., antibodies TIM3.2 toTIM3.18 may have any desired constant region, e.g., those shown in FIG.13.

CDRs and variable domains were expressed in the context of aneffectorless IgG1 constant region (allotype “f”), which comprises thesubstitutions L234A, L235E, G237A, A330S and P331S (“IgG1.f”) andIgG1.3f, an effectorless IgG1 constant region (allotype “f”), whichcomprises substitutions L234A, L235E, G237A, i.e., it differs from IgG1.If only in not having the A330S and P331S substitutions. The CDRs andvariable regions may also be used in the context of IgG4, e.g., IgG4P(i.e., IgG4 with a “S228P” substitution). Certain CDRs and frameworkregions of these antibodies have also been mutated. Specifically, VHCDR2of 13A3 and 8B9, VHCDR3 of 13A3 and VHFR4 have been mutated. A list ofIgG1.1f and IgG1.3f antibodies that have been produced and otherantibodies that can be made is provided in FIG. 13, Table 1 and in thesequence listing. Antibodies expressed recombinantly include thosedescribed in the Examples below, as well as antibodies 3G4, 8C4, 9F6,8B9, 17C8, 5D6 that have been expressed as IgG1.f antibodies.

A sequence alignment of the heavy and light chain variable regions ofantibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 is provided in FIGS.8A and 9A, respectively. The VH and VL region sequence designation areprovided in FIGS. 8B and 9B, respectively. A sequence alignment of thewildtype and mutated 13A3 VH chains is provided in FIG. 10. A sequencealignment of the wildtype and mutated 9F6 VH chain is provided in FIG.11. A sequence alignment of the wildtype and mutated 8B9 VH chains isprovided in FIG. 12.

Example 2: Characterization of the Human Anti-TIM3 Antibodies

The selected anti-TIM3 antibodies were assayed for binding toTIM3-expressing cells. FIG. 14A shows the binding of various anti-TIM3antibodies to cells transfected with human TIM3 (FIG. 14A), and toanti-CD3/anti-CD28-activated human T cells (FIG. 14B), as determined byflow cytometry. The antibodies were also tested for binding to cyno TIM3by using cells transfected with cyno TIM3 (FIG. 15A) andanti-CD3/anti-CD28-activated cyno T cells (FIG. 15B). FIG. 15A showsthat 13A3 has the best binding EC₅₀ for cyno-TIM3 transfected cell line,and it is the only anti-TIM3 antibody that is reactive with activatedcyno T cells.

Example 3: Binding Affinity of TIM3 Antibodies to Human and Cyno TIM3Determined by Surface Plasmon Resonance

Kinetics and affinity of anti-TIM3 13A3 Fab fragments towards human andcyno TIM3 were determined on a Biacore T200 instrument at 37° C. in PBSpH 7.4 supplemented with 0.05% (v/v) Tween-20, as further describedbelow. The human TIM3 protein used consisted of the extracellular domain(ECD) of human TIM3 linked to a mouse Fc, thereby forming a dimerichTIM3 ECD-Fc protein (“hTIM3-mFc”). This fusion protein was expressedfrom stably transfected CHO cells, and purified out of the medium usingprotein A affinity, followed by size exclusion chromatography. Therecombinant cynomolgus TIM3 protein used consisted of the extracellulardomain of cynomolgus TIM3 followed by linker and affinity tags, therebyforming a monomeric cynoTIM3 ECD protein (“cyno TIM3-MycHisAvi”). Thisfusion protein was expressed from transiently transfected Expi293 cells(Life Tech) and the protein was isolated from the medium and purifiedout using affinity tag (6×His), followed by size exclusionchromatography.

The amino acid sequence of hTIM3-mFc was as follows:

(SEQ ID NO: 375) SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGASVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHT EKSLSHSPGK

The amino acid sequence of cynoTIM3-MycHisAvi was as follows:

(SEQ ID NO: 376) SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKSPGGGSGGGSEQKLISEEDLGHHHHHHGLNDIFEAQKI EWHE

Fabs of 13A3 and TIM3.18.IgG1.3 linked to a histidine tail were used.The amino acid sequence of 13A3 Heavy Chain (HC) Fab 6×His was asfollows:

(SEQ ID NO: 365) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGHHHHHH

The amino acid sequence of 13A3 Heavy Chain (HC) N60Q D101E Fab 6×Hiswas as follows:

(SEQ ID NO: 366) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGHHHHHH

Recombinant 13A3 and TIM3.18.IgG1.3 Fabs were made using transienttransfection of Expi293 (Life Tech). The expressed Fab comprised theheavy chain variable region followed by the CH1 of hIgG1, and lightchain variable region followed by the CL domain of hKappa. The expressedFab was secreted into the medium and purified using affinity tag(6×His).

An anti-mouse antibody capture chip was prepared on a Biacore CM4 seriesS chip (GE Healthcare Life Sciences catalog #BR-1005-34) using theBiacore capture kit for mouse antibodies (catalog #BR-1008-38). HumanTIM3-mouse Fc fusion protein was captured on flow cells 2 and 3 in twodifferent surface densities. Cyno TIM3-mouse Fc fusion protein wascaptured on flow cell 4. Flow cell 1 (blank capture surface) served as areference. Recombinantly expressed, His-tagged antibody Fab fragmentswere flowed as analytes over all surfaces in a 3-fold, 6-membereddilution series with 1.0 μM top concentration and 4.1 nM bottomconcentration. Resulting sensorgrams were double-referenced (using flowcell 1 and a buffer blank) and fitted to a 1:1 Langmuir binding modelwith mass transport. Data from flow cells 2 and 3 were fitted globally.

The rate of complex formation (K_(a)) and dissociation (K_(D)) as wellas overall dissociation constant (K_(D)) are provided in Table 2.

TABLE 2 Kinetics and affinity of binding of anti-TIM3 antibodies 13A3and TIM3.18.IgG1.3 to human and cyno TIM3 proteins Ligand Analytes ka(1/Ms) kd (1/s) K_(D) (nM) Human TIM3 hTIM3Fc/13A3 Fab 3.2 × 10⁶ 6.9 ×10⁻³ 2.2 Cyno TIM3 cynoTIM3Fc/13A3 Fab 2.4 × 10⁶ 5.3 × 10⁻² 22 HumanTIM3 hTIM3Fc/TIM3.18 Fab 3.2 × 10⁶  5 × 10⁻³ 1.6 Cyno TIM3cynoTIM3Fc/TIM3.18 Fab 3.4 × 10⁶ 5.9 × 10⁻² 17

The experiments with 13A3 were not conducted on the same day as thosewith TIM3.18.

Example 4: Binding Affinity of TIM3 Antibodies to Human and Cyno TIM3Determined by Scatchard Analysis

TIM3.18.IgG1.3 antibody was radioiodinated with ¹²⁵I—Na (1mCi;PerkinElmer Catalog NEZ033H001 MC) using IODO-GEN® solid phaseiodination reagent (1,3,4,6-tetrachloro-3a-6a-diphenylglycouril; PierceCatalog 28601). Excess iodide was removed using a desalting column(Pierce Catalog 43243). Fractions of labeled antibody were collected andanalyzed for radioactivity on a Wizard 1470 gamma counter (PerkinElmer).The ¹²⁵I-TIM3.18.IgG1.3 antibody concentration in each fraction wascalculated with the Qubit fluorometer from Invitrogen. Radiopurity wasestablished by thin layer chromatography of peak protein and radioactivefractions (Pinestar Technology Catalog 151-005).

Radio iodinated TIM3.18.IgG1.3 antibody binding to CHO cells expressinghuman or cyno TIM3 was demonstrated by incubating the CHO cellsexpressing human or cyno TIM3 with a titration of ¹²⁵I-TIM3.18.IgG1.3antibody. Nonspecific binding was determined by binding in the presenceof a titration of a 100 fold molar excess of unlabeled antibody and wassubtracted from total CPM to calculate specific binding. A linearstandard curve of ¹²⁵I-TIM3.18.IgG1.3 antibody concentration versus CPMwas used to extrapolate specific activity, maximal nM bound¹²⁵I-TIM3.18.IgG1.3 antibody and thereby calculate receptor number percell.

The results are shown in FIGS. 27A and 27B. The ¹²⁵I-TIM3.18.IgG1.3antibody standard curve (FIG. 27A) shows that 1 nM of ¹²⁵I labeledantibody equals 81119.3 cpm. The number of receptors per cell iscalculated by the following equation: (Bmax)× (Avogadro's number)×(AssayVolume)/# of cells per well. The results show that the TIM3.18.IgG1.3antibody has an affinity of 0.26-0.48 nM for overexpressed human TIM3 onCHO cells (having 414,720 receptors per cell) and an affinity of0.36-0.48 nM for overexpressed cyno TIM3 (having 235,944 receptors percell).

A similar analysis conducted with ¹²⁵I-TIM3.18.IgG1.3 antibody onactivated human Th1 cells from 2 donors (50,000 cells/well) provided anaffinity of 0.125-0.164 nM, despite an almost four fold difference innumber of receptors per cell between donors (FIG. 28). Radio iodinatedTIM3.18.IgG1.3 binding to human TIM3 was demonstrated by incubatingactivated primary human Th1 cells (prepared as described in otherExamples herein) with a titration of ¹²⁵I-TIM3.18.IgG1.3. Nonspecificbinding was determined by binding in the presence of a titration of a100 fold molar excess of unlabeled antibody and was subtracted fromtotal CPM to calculate specific binding. A linear standard curve of¹²⁵I-TIM3.18.IgG1.3 concentration versus CPM was used to extrapolatemaximal nM bound ¹²⁵I-TIM3.18.IgG1.3 and thereby calculate receptornumbers per cell.

Example 5: Lack of Cross-Reactivity of TIM3.18.IgG1.3 to Human TIM1,Human TIM4 and Mouse TIM3

Upon a blast search of the TIM-3 IgV domain against the entire genebank, the highest homologous molecules were TIM1 and TIM4 (45%identity). Selectivity profiling of TIM3.18.IgG1.3 using human TIM1 orTIM4-transfected cell lines by flow cytometry showed no cross-reactivityto TIM1 or TIM4. It was also shown by flow cytometry on mouse TIM3transfected cells, that TIM3.18.IgG1.3 is not cross-reactive with mouseTIM-3 transfected cells.

Example 6: IFN-γ Production by Tumor Infiltrating Lymphocytes (TILs) isEnhanced by Anti-TIM3 Antibodies

To characterize the anti-TIM3 antibodies further and identify those thatare more likely to have significant T cell stimulating activity in vivo,a specific T cell assay was developed. The assay measures the amount ofIFN-γ secreted from tumor infiltrating lymphocytes (TILs), isolated fromfresh tumor tissue, and incubated in the presence of irradiated CHOcells, expressing CD3 (“CHO-OKT3 cells”), in the presence or absence ofa TIM3 antibody (or control). Without wanting to be limited by aspecific mechanism of action, secretion of IFN-γ in the presence of agiven anti-TIM3 antibody indicates that the antibody inhibits thenegative signaling normally provided by TIM3 on the TILs, and stimulatesactivation (i.e., IFN-γ production) of the TILs.

Fresh tumor tissue (including tumor infiltrating lymphocytes (TILs))from a renal cell carcinoma patient was prepared into a single cellsuspension by enzymatic digestion (Miltenyi, Catalog #130-095-929). Thecell viability was more than 80%, as determined by FACS. 1.5 10⁵ cellswere co-cultured for 5 days with 2.5 10⁴ irradiated (67,000 RAD for 1 hr20 min; Rad Source Irradiator, RS-2000 Biological System) CHO-OKT3 cellsin IL-2-containing medium (IL-2 (Peprotech, Catalog #200-02) at 20IU/ml) in the presence of either an isotype control antibody oranti-TIM3 antibody at different concentrations. At day 5 of the culture,the cell supernatant was collected and the IFN-γ level was assessed byELISA (BD Opteia hIFNy ELISA kit, BD, Catalog #555152). The results,which are shown in FIG. 16, indicate that the anti-TIM3 antibodies 13A3,3G4, 17C3, 17C8 and 9F6 stimulate IFN-γ production by renal cellcarcinoma TILs.

Fresh tumor tissue from a lung cancer patient was digested with aMiltenyi enzymatic digestion kit (Miltenyi, Catalog #130-095-929). Thesingle cell suspension was co-cultured with irradiated (67,000 RAD for 1hr 20 min; Rad Source Irradiator, RS-2000 Biological System) CHO-OKT3cells in IL-2-containing medium (IL-2 (Peprotech, Catalog #200-02) at 20IU/ml in the presence of an isotype control antibody or anti-TIM3antibody at different concentrations. At day 5 of the culture, the cellsupernatant was collected for IFN-γ ELISA (BD Opteia hIFNγ ELISA kit,BD, Catalog #555152). The results, which are shown in FIG. 17A, indicatethat the anti-TIM3 antibodies tested (i.e., 13A3 and 3G4) stimulateIFN-γ production by lung cancer TILs.

In addition, at day 3.5 of a co-culture of the cell suspension from thelung cancer tumor tissue with irradiated (67,000 RAD for 1 hr 20 min;Rad Source Irradiator, RS-2000 Biological System) CHO-OKT3 cells treatedwith an isotype control antibody or anti-TIM3 antibody in the presenceof IL-2, cells were incubated with BD GolgiStop overnight. Subsequently,the cells were first stained with cell surface markers, CD45, CD4, CD8,TIM3 and PD 1, and then fixed and permeabilized with BD Cytofix/Cytopermkit followed by intracellular IFN-γ staining. The results, which areshown in FIG. 17B, show that the percentage of intracellular IFN-γexpressing cells is increased in CD8⁺ cells (lower panel) upon anti-TIM3antibody treatment.

FIG. 18 shows the pooled data from multiple tumor TIL experiments(performed as described above in this Example) in response to anti-TIM-3antibodies clones 13A3 or 3G4 (i.e., every dot on the figure representsTILs from one patient tumor sample treated with either 13A3 or 3G4).Several renal cell carcinoma (RCC) and lung cancer TILs responded toanti-TIM-3 antibody in promoting IFN-γ production, while a single TILpreparation from a thyroid tumor failed to do so.

Example 7: FACS Based Cross-Blocking of Anti-TIM3 Antibodies

Total human T cells were isolated from PBMC using a Miltenyi T cellpurification kit and activated with plate-bound anti-CD3 (1 μg/ml;Anti-CD3 clone OKT3, eBioscience, Catalog #16-0037-85) and solubleanti-CD28 (1 μg/ml; Anti-CD28 clone CD28.2, BD Biosciences, Catalog#555725) for 4 days. TIM3 was expressed in >80% of T cells, asdetermined by FACS. The T cells were incubated with various anti-TIM3antibodies for 30 minutes, followed by incubation with selectedbiotin-labeled anti-TIM3 antibodies for 30 minutes and detected byPE-conjugated streptavidin. The results, which are shown in FIG. 19,indicate that antibodies 13A3, 3G4, 17C3, 17C8, and 9F6 are in the samebinning group (Group I), i.e., cross-compete each other, whileantibodies 8B9 and 8C4 are in a separate binning group (Group II), i.e.,do not cross-compete with the antibodies in Group I, but cross-competewith each other. The antibodies in binning group I were shown to havebiological activity (see Examples), while those in binning group II hadweaker activity. Two anti-TIM3 antibodies which did not cross-competewith either Group I or Group II, did not appear to have any biologicalactivity. The antibodies of binning group I were also those thatinterfered with TIM3 binding to PS (as further described herein).

Example 8: Epitope Mapping by Yeast Surface Display Method

The nucleotide sequence encoding the extracellular domain of human TIM3(NM 032782), i.e.,SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLAD SGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIG, (SEQ ID NO: 290) wascloned into the yeast display plasmid PDV0023 by ligation into the XhoIand NotI restriction enzyme sites. Low rate random mutagenesis wasperformed on the sequence to generate single point mutations across theTIM3 coding region using the GeneMorph II Random Mutagenesis kit fromAgilent Technologies. A library of 9.8×10⁶ clones was generated in VWK18gal S. cerevisiae cells. 2×10⁸ library cells were passaged and inducedfor antibody labeling and cell sorting. About 2×10⁷ induced cells wereincubated with 100 nM a primary target anti-human TIM3 antibody and 100nM anti cMyc (9E10) antibody for 1 hr at 25° C. Cells were washed thendetected with fluorescently labeled goat anti human IgG-PE and goatanti-mouse IgG-A633 secondary antibodies for 45 min at 4° C. to detectthe bound primary antibodies on the cell surface. Labeled cells weresorted on a BD FACSARIA II instrument into yeast culture media. Cellsthat were positively labeled with anti-Myc antibody and negativelylabeled with anti-human TIM3 were collected. The APC+/PE− population ofcells were expanded, passaged and induced for a second round ofidentical labeling and sorting to enrich the desired populations. Yeastplasmid DNA was purified out of about 2 10⁷ cells from the unselectedlibrary and both rounds of selected, sorted cells. For each cellpopulation the TIM3 target sequence was rescued and purified out of theyeast plasmid DNA by PCR using vector specific primers that flank thehuman TIM3 sequence. The target sequence PCR products were subjected toNGS library preparation using the Nextera XT DNA Library kit forIllumina Sequencing from Illumina. The prepared libraries were sent toEA/Q2 Solutions for high throughput sequencing on the MiSeq platformfrom Illumina with 300 cycles/flow cell. Between 0.5 and 1.0 millionsequence reads for each library were compared to the wild type TIM3sequence, and mutations at each position along the sequence weretabulated. The difference in mutational frequency at each residueposition between the selected rounds and the unselected library werecalculated and used to determine critical residues for antibody binding.Positions with high mutation frequency were examined for surfaceexposure using a human TIM3 structural model based on known crystalstructures of mouse TIM3 (PDB: 2OYP, and PDB: 3BIB). High mutationfrequency, surface exposed residues are considered part of epitopes,while high mutation frequency, buried residues are considered as falsepositives. False positive residues are usually those that disrupt eitherlocal or core folding of the protein, and indirectly alter binding ofthe Ab to its surface epitope.

FIG. 20 shows the residues that were determined to be part of theepitope on human TIM3 for each of the antibodies used. In addition, D104shows positive mutational score, in all mappings, and may be astructural salt bridge to R81. For the 8B9 epitope, L84 shows highmutation frequency, though appears buried in the structure supportingthe epitope residues. Q113 shows a low, but positive score for 13A3. Itlikely plays an epitope region structural supporting role, but has somesurface exposure.

Example 9: Blocking of TIM3-PtdSer Interaction by TIM3 Antibodies

The “tandem blocking assay” shown in FIG. 21A was used to determinewhether the anti-TIM3 antibodies inhibit the interaction between humanTIM3 and phosphatidylserine (“PtSer” or “PS”). Since PS is not watersoluble, PS-liposome was made for the assay. Briefly, lipids were mixedwith methanol/chloroform and then chloroform was evaporated undernitrogen stream and vacuum overnight. Subsequently, the lipids weresonicated with micro tip to fully disperse lipid to create liposome.They were further passed through an extruder >10 times to ensurehomogenous size.

PS liposome are generated with PS (L-α-phosphatidylserine (Brain,Porcine) Avanti Polar Lipids Cat#840032C) suspended in chloroform. PSstock is first diluted in chloroform to the necessary amount, and thechloroform is evaporated under a nitrogen stream until no liquid isvisible. To remove trace amounts of chloroform, dried PS is placed undervacuum overnight. Dried PS is then suspended in PBS via vortex and briefsonication until the solution turns cloudy. To create size defined PSliposomes, an extruder with a 100 nm filter is used. Suspended PS isloaded into the extruder and passed through the filter at least 10times. At this point the PS liposome is diluted in PBS to the neededconcentration.

In the “tandem blocking assay”, TIM3 (ECD)-Fc was captured on Octetbiosensor, and anti-TIM3 antibody and PS-liposomes were allowed to bindto the TIM3 protein. When anti-TIM3 binds to a region that is blockingPS binding, PS-liposome shows no binding.

The results, which are shown in FIG. 21B, indicate that antibodies 3G4,13A3, 17C3, and 17C8 inhibit binding of PtSer to human TIM3, whereas 2other anti-TIM3 antibodies, i.e., AbA and AbB, do not inhibit binding ofPtSer to human TIM3. As further described in the Examples, theantibodies that inhibit PtSer binding are also those who have thestrongest functional activity (as determined in the Th1 and TIL assays).

Example 10: HDX-MS Epitope Mapping of TIM3 Antibodies

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was utilized toprobe binding epitopes of hTIM-3 with antibodies 13A3 and 3G4.

HDX-MS probes protein conformation and conformational dynamics insolution by monitoring the rate and extent of deuterium exchange ofbackbone amide hydrogen atoms [1, 2]. The level of HDX depends on thesolvent accessibility of backbone amide hydrogen atoms and the proteinhydrogen bonds. The mass increase of the protein upon HDX can beprecisely measured by MS. When this technique is paired with enzymaticdigestion, structure features at the peptide level can be resolved,enabling differentiation of surface exposed peptides from those foldedinside, or from those sequestered at the interface of a protein-proteincomplex. Typically, the deuterium labeling and subsequent quenchingexperiments are performed, followed by enzymatic digestion, peptideseparation, and MS analysis.

Prior to epitope mapping experiments, non-deuteriated experiments werecarried out to generate a list of common peptides for recombinant humanTIM-3 ((hTIM3-ECD (22-200) His-tagged (see FIG. 25); 10 μM, SinoBiological Inc.) and protein complexes of hTIM-3 with Fab of antibodies13A3 and 3G4 (1:1 molar ratio). The samples were injected into WatersEnzymate BEH pepsin enzyme column (2.1×30 mm), and digested for 3 min at200° C. The cooling chamber of the UPLC system, which housed all thechromatographic elements, was held at 0.0±0.1° C. for the entire time ofthe measurements. The injected peptides were trapped and desalted for 3min at 100 μL/min and then separated in 6 min by a 5-40%acetonitrile-water gradient at 65 μL/min. The separation column was a1.0 mm×50.0 mm ACQUITY UPLC BEH C18 column (Waters). Identification ofthe peptic peptides was accomplished through a combination of exact massanalysis and MSE using ProteinLynx Global SERVER 2.5 (Waters) on WatersHDX-MS system.

In the HDX-MS experiment, 5 μL of each sample (hTIM-3 or hTIM-3 with Fabof antibody 13A3 or 3G4) was diluted into 55 μL of D₂O buffer (10 mMphosphate buffer, D₂O, pH7.0) to start the labeling reactions. Thereactions were carried out for different periods of time: 1 min, 10 minand 240 min. By the end of each labeling reaction period, the reactionwas quenched by adding quenching buffer (100 mM phosphate buffer with 4MGdnCl and 0.4M TCEP, pH 2.5, 1:1, v/v). 50 μL of quenched sample was wasdigested online using the same conditions as in non-deuteriatedexperiments. All comparison experiments were performed under identicalexperimental conditions. All experiments were performed in duplicate.The resulting relative deuterium levels were plotted versus the exchangetime with use of the software program DynamX 3.0™ (Waters).

As shown in FIG. 25, sequence coverage of 97.3% of hTIM-3 was obtainedin HDX-MS experiments. As shown in FIG. 26, HDX-MS data analysis ofhTIM-3 upon binding with Fab of antibodies 13A3 and 3G4 identified thefollowing discontinuous epitopes:

mAb 13A3: ⁴⁹VPVCWGKGACPVFE⁶² (SEQ ID NO: 367), ¹¹¹RIQIPGIMNDEKFNLKL¹²⁷(SEQ ID NO: 368), and fragment ¹¹⁹NDEKFNLKL¹²⁷ thereof; and

mAb 3G4: ⁴⁰YTPAAPGNLVPVCWGKGACPVFE⁶² (SEQ ID NO: 369), ⁶⁶VVLRTDERDVNY⁷⁷(SEQ ID NO: 370), ⁷⁸WTSRYWLNGDFRKGDVSL⁹⁵ (SEQ ID NO: 371), and¹¹⁰CRIQIPGIMNDEKFNLKL¹²⁷ (SEQ ID NO: 372).

FIG. 26 shows the HDX-MS peptides to which antibodies 13A3 and 3G4 bind,as determined using the HDX-MS protocol described in this Example.

Thus, antibody 13A3 interacts with regions of amino acid residues 49-62and 111-127 of hTIM3, but does not significantly interact with otherregions, such as the region that is N-terminal to amino acid residue Y40or V49, the region that is located between amino acid residues E62 andR111, and the region that is C-terminal to amino acid residue L127. 13A3binds to the phosphatidylserine binding loop of the TIM-3 IgV domain.

Example 11: On-Target IHC Staining by TIM3.18 in Human TissueCross-Reactivity

Immunohistochemistry (IHC) was performed with 13A3 on frozen sections ofhuman and cynomolgus monkey spleen. In both species, 13A3 (0.5 μg/mL)stained the endothelium of venous sinusoids. As expected, antibody 3G4,which does not cross-react with cynomolgus TIM-3, stained the humanspleen but not the cynomolgus spleen.

In preliminary tissue cross-reactivity analysis, FITC-conjugatedTIM3.18.IgG1.3 was applied to frozen sections or smears from 20 types ofnormal human tissues, which include cerebrum, cerebellum, heart, liver,lung, kidney, PBMC smears, spleen, tonsil, thymus, skin, colon, smallintestine, stomach, pancreas, peripheral nerve, pituitary, thyroid,prostate, and placenta (1 donor each). Specific staining was observed ina subset of mononuclear cells (MNC) in PBMC, spleen, and tonsil, as wellas in epithelial reticular cells or macrophages in the thymus. The mostprofound staining was in macrophage/DC-like cells, which were observedin every tissue examined, including tissue-specific macrophages (e.g.,Kupffer cells in the liver, dermal macrophages/DC in the skin, andHofbauer cells in the placenta). At the organ level, the strongeststaining was found in the spleen. Besides small subsets of MNC, strongstaining was very frequently seen in splenic endothelial cells in thered pulp. In addition, positive staining was observed in a small subsetof cortical tubular epithelial cells in the kidney cortex.

Example 12: Anti-Tumor Activity of Combined Anti-TIM3 and PD-1Antibodies in Mice

Rat anti-mouse TIM-3 (RMT3 23) and PD-1 (RMP1-14) commercial antibodies(Bio-X-Cell) were evaluated in a CT26 colorectal tumor model. Theexperimental design was similar to a previously described in vivo studyNgiow et al. (2011) Cancer Res. 71:3540. Since TIM-3 is expressedrelatively late (Day 15) in this tumor model, a small volume of tumorcells (2×10⁵) was implanted in the flank of each mouse so that tumorgrowth would be minimal, allowing time for TIM-3 expression. When tumorsbecame palpable at Day 8, mice were randomized into 4 treatment groupsof 10 mice each, with a mean tumor volume of 40 mm³. RMT3-23 (anti-TIM-3antibody) and RMP1-14 (anti-PD-1 antibody) were administered byintraperitoneal injection, either as single or combined agents (250μg/per injection of each antibody); the isotype control was administeredat 500 μg/per injection. Each study animal received 250 μg of oneantibody or 500 μg of 2 combined antibodies for each injection and for atotal of 3 doses. Tumor size was assessed biweekly. Mice in each groupreceiving single or combined test articles exhibited antitumor activity,with 2/10 mice in the anti-PD-1 monotherapy group and 6/10 mice in thecombined anti-PD-1 and anti-TIM-3 group remained tumor-free at studytermination (FIG. 24A). A previous CT26 study of the same designproduced similar results, with 3/10 mice in the anti-PD-1 monotherapygroup and 7/10 mice in the combined anti-PD-1 and anti-TIM-3 grouptumor-free. There was little or no antitumor activity with anti-TIM-3administered as a single agent.

Of note, the EC50 value of RMT3-23 binding to activated mouse T cells is1.7 nM, which is 17-fold weaker than the EC50 of TIM3.18 for binding tohuman TIM-3. Another rat anti-mouse TIM-3 antibody, (Ab M), whichcross-blocks RMT3-23, has an EC50 of 0.1 nM in binding to activatedmouse T cells, which is equivalent to the EC50 of TIM3.18. Like RMT3-23,Ab M maps to the PS-binding loops of mouse TIM-3. Use of this antibodywith a mIgGi-D265A (Fc-inert isotype) heavy chain constant region in theCT26 tumor model demonstrated that it enhanced the antitumor response toanti-PD-1 (FIG. 24B).

Example 13: Th1 Cell Proliferation Assay with TIM3 Antibody (Full Lengthor Fab) Blockade

To further characterize the anti-TIM3 antibodies, a specific T cellproliferation assay using in vitro polarized Th1 cells was developed.Polarized Th1 cells were obtained by repeatedly restimulating naïve CD4+T cells. These cells were then incubated with irradiated (growtharrested) CHO-OKT3 cells in the presence of anti-TIM3 antibodies (orcontrol) and Th1 cell proliferation was measured.

Naïve CD4 T cells were polarized to Th1 memory-like T cells as follows.Naïve CD4 T cells were purified from PBMCs using a naïve CD4 T cellisolation kit from Miltenyi. The cells were cultured for 3-4 days inIMDM/10% FBS at 3.6×10⁵ cells/ml in the presence of: CD3/CD28 coated(80%/20% respectively) beads at 1 bead to 1 cell ratio; 10 ng/ml humanIL-2; 1 ng/ml human IL-12 and 10000 ng/ml anti-human IL-4 antibody.After the incubation, the cells were collected in a tube, the beads wereremoved with a magnet and the cells were returned to culture in a newflask. Recombinant human IL-2 was added to a final concentration of 4ng/ml, and the cells were incubated for an additional 3 days. The cellswere then collected and washed with 1×IMDM/10% FBS. The cells werecounted, resuspended in IMDM/10% FBS at 4.1×10⁵ cells/ml, and culturedfor 3-4 days in the presence of: CD3/CD28 coated (80%/20% respectively)beads at 1 bead to 1 cell ratio; 10 ng/ml human IL-2; 1 ng/ml humanIL-12 and 10000 ng/ml anti-human IL-4 antibody. After the incubation,the cells were collected in a tube, the beads were removed with a magnetand the cells were returned to culture in a new flask. Recombinant humanIL-2 was added to a final concentration of 4 ng/ml, and the cells wereincubated for an additional 2-3 days. The polarized Th1 cells were thenharvested and washed 3 times. On the day of assay set-up, the polarizedTh1 cells were re-suspended in complete medium.

The following reagents were used:

Dynabeads M-450 Epoxy Dynal Biotech ASA 140.11 100 mM Sodium PhosphateBuffer, Teknova 0214-250 pH 8.5 Functional Grade anti-hCD3 CloneeBioscience 16-0038-85 UCHT-1 Functional Grade anti-hCD28 CloneeBioscience 16-0289-85 CD28.2 Recombinant Human IL-2 PeproTech, Inc.200-02 Recombinant Human IL-12 PeproTech, Inc. 200-12 anti-human IL-4eBioscience 16-7048-85 Iscove's DMEM Mediatech, Inc. 10-016-CM FetalBovine Serum (heat-inactivated) Hyclone SH30071.03

The CHO-OKT3 cell line was grown in shaker flasks and irradiated (67,000RAD for 1 hr 20 min; Rad Source Irradiator, RS-2000 Biological System)on the day of assay set-up. The irradiated CHO-OKT3 cells provided Tcell stimulation and exposed phophatidylserine (PS) as confirmed byAnnexin V staining.

TIM3.18.IgG1.3 or isotype control was titrated from 20 μg/mL by 4-foldserial dilutions, with each condition set up in triplicate.TIM3.18.IgG1.3 Fab was titrated from 53 μg/mL also by 4-fold serialdilution. The TIM3.18.IgG1.3 Fab fragment was the same as that used inthe crystallography experiment (see Examples).

The cultures were set up in flat-bottom TC-treated 96-well plates(Costar) with 1×10⁵ polarized Th1 cells and 2.5×10⁴ irradiated CHO-OKT3cells (CHO:T cell ratio of 1:4) in 200 μL complete medium per well inthe presence of 0.1 μg/ml anti-CD28 (clone CD28.2, BD Biosciences,Catalog #555725), and incubated for 3 days at 37° C. and 5% CO₂. Theplates were then pulsed with 1 μCi tritiated thymidine (Perkin Elmer,Catalog # NET027001MC) per well for 16 hours and then the cells wereharvested onto filter plates (Perkin Elmer) for analysis of tritiatedthymidine incorporation in order to assess proliferation.

The results, which are shown in FIGS. 29A and 29B, indicate that theanti-TIM3 antibody TIM3.18.IgG1.3 increased Th1 cell proliferation in adose-dependent manner in the CHO-OKT3/Th1 co-culture cell assay. Theoverall activity of TIM3.18.IgG1.3 is equivalent to that of its parentalantibody, 13A3 (IgG4 isotype) (FIG. 29A). TIM3.18.IgG1.3 Fab fragmentalso exhibited a dose-dependent induction of proliferation (FIG. 29B) inthe CHO-OKT3/Th1 cell assay.

Thus, TIM3.18.IgG1.3 (both full length and Fab) potentiated Th1 cellactivity in a dose-dependent manner in co-culture with irradiatedCHO-OKT3 cells. The presence of activity with the Fab fragment indicatedthat TIM3.18.IgG1.3 works as an antagonistic antibody and that TIM-3 isan inhibitory receptor for T cell function. No Fc cross-linking wasrequired for TIM3.18.IgG1.3 biological activity.

Example 14: Th1 Cell Proliferation Assay with TIM 3 and PD 1 Co-Blockade

This assay was a co-culture between irradiated (growth arrested; 67,000RAD for 1 hr 20 min; Rad Source Irradiator, RS-2000 Biological System)CHO-OKT3 cells transfected with human PD-L1 (CHO-OKT3-PD-L1), and Th1cells at a CHO:T cell ratio of 1:4 in the presence of anti-CD28. TheCHO-OKT3-PD-L1 cell line was grown in shaker flasks and irradiated onthe day of assay set-up. The polarized Th1 cells were prepared asdescribed in the other Examples described herein. On the day of assayset-up, the polarized Th1 cells were re-suspended in complete medium.

Anti-PD-1 antibody nivolumab was titrated from 10 μg/mL by 10-foldserial dilutions, with each condition set up in triplicate. TIM-3antibody TIM3.18.IgG1.3 or isotype control was spiked in at 20 μg/mL.

The cultures were set up in flat-bottom TC-treated 96-well plates(Costar) with 1×10⁵ Th1 cells and 2.5×10 CHO-OKT3-PD-L1 cells in 200 μLcomplete medium per well [in the presence of 0.1 μg/ml anti-CD28 (cloneCD28.2, BD Biosciences, Catalog #555725), and incubated for 3 days at37° C. and 5% CO₂. The plates were then pulsed with 1 μCi tritiatedthymidine (Perkin Elmer, Catalog # NET027001MC) per well for 16 hoursand then the cells were harvested onto filter plates (Perkin Elmer) foranalysis of tritiated thymidine incorporation in order to assessproliferation.

The results, which are shown in FIG. 30, indicate that anti-PD-1antibody nivolumab increased proliferation of Th1 T cells stimulatedwith CHO-OKT3-PD-L1 cells in a dose-dependent manner, and that theproliferation was greatly enhanced in combination with TIM3.18.IgG1.3.Co-blockade of TIM-3 and PD-1 pathways showed additive effect in thisassay.

Example 15: Tumor-Infiltrating Lymphocyte IFN-γ Release Assay withTIM3.18.IgG1.3 Blockade

For this assay, fresh tumor tissues were obtained from a surgicallyremoved human renal cell carcinoma sample or breast cancer sample. Thetumor-infiltrating lymphocytes (TIL) were isolated using an enzymaticdissociation kit (Miltenyi, Catalog 130-095-929). TILs were supplementedwith 20 IU/mL IL-2 (Recombinant human IL-2, Peprotech, Catalog 200-02)and co-cultured with irradiated (growth arrested; 67,000 RAD for 1 hr 20mmin; Rad Source Irradiator, RS-2000 Biological System) CHO-OKT3 cellsat a CHO:T ratio of 1:6. The CHO-OKT3 cell line was grown in shakerflasks and irradiated on the day of assay set-up.

TIM-3 antibody TIM3.18.IgG1.3 or isotype control was titrated from 20μg/mL by 4-fold serial dilutions, with each condition set up intriplicate. The cultures were set up in flat-bottom TC-treated 96-wellplates (Costar) with 1.5×10⁵ T cells and 2.5×10⁴ irradiated CHO-OKT3cells in 200 μL per well in IMDM+5% FBS and 5% human AB serum (Gemini,Catalog #100-512), and incubated for 5 days at 37° C. and 5% CO₂. Thesupernatant was harvested from each sample for IFN-γ measurement byELISA (BD Opteia hIFN-γ ELISA kit, BD, Catalog 555152).

The results, which are shown in FIG. 31, for the renal cell carcinomaTILs, and in FIG. 32, for the breast cancer TILs indicate thatTIM3.18.IgG1.3 increased IFN-γ production in a dose-dependent manner inthe CHO-OKT3/TIL co-culture assay, with up to 4-fold increase overnegative controls at higher concentrations of TIM3.18.IgG1.3 in therenal cell carcinoma TIL assay.

Example 16: TIM3.18.IgG1.3 Promotes IFN-γ Secretion in M0:T AllogeneicMLR Assay

Isolated CD14+ monocytes from healthy donors were differentiated to theM0 stage in culture medium containing M-CSF. After Day 6 in culture, asignificant population of macrophages were expressing CD163+ and CD206+on the cell surface by FACS staining, consistent with the signature ofsuppressive macrophages. By flow cytometry with an anti-TIM-3 antibody,TIM-3 was shown to be expressed in the M0 macrophages (FIG. 33). TheseM0 macrophages were then irradiated (5,000 RAD for 7 min; Rad SourceIrradiator, RS-2000 Biological System) and co-cultured with an allogenicdonor's total T cells, and at Day 6 post-co-culturing, the mixed cellswere pulsed with ³H-thymidine overnight for assessing T cellproliferation.

The results, which are shown in FIG. 34, indicate that TIM3.18.IgG1.3increased T cell proliferation as compared to isotype control.

Example 17: Crystal Structure of TIM3.18.IgG1.3 Fab Interacting withhTIM3

hTIM3 IgV region was co-crystallized with a Fab fragment of TIM3.18 asfollows. The sequences used were the following:

hTim3_IgV: (SEQ ID NO: 377; TIM3 sequence is underlined)HHHHHHSAALEVLFQGPGSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPA Tim3.18_Fab: (SEQ ID NO: 366)QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGHHHHHH Tim3.18_kappa: (SEQ ID NO: 29)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

Expression and purification. A histidine tagged hTim3 IgV domain wasexpressed in E. coli (BL21 DE3) with a pET47b vector. Purification andrefolding was done following published protocol for mTim3 (DeKruyff etal. J. Immunology 2010). Tim3.18 Fab was transiently expressed in HEK293cells, and purified via the C-terminal His Tag on the heavy chain.

Crystallization of complex and structure determination. The crystalstructure of hTim3 IgV domain with Tim3.18 Fab was resolved to 1.5 Å.The Fab:antigen complex was first screened for cyrstallizationconditions with various screens from Hampton Research, and crystalsclusters were observed in conditions with PEG 3350 with pH ranging from6.5 to 5.5. The crystal growth condition was further optimized to allowthe growth of single crystals. Single crystals were harvested withglycerol as the cryoprotectent, and flash frozen in liquid nitrogen.Data collection was conducted at IMCA-CAT at APS using Pilatus-6Mdetector. Diffraction images were processed with Global Phasingsoftware, and phased using a Fab model of Tim3.18. Multiple rounds ofrefinement were done using CCP4 suite, Coot, Phenix, and Global Phasingsuite of software.

The resolved hTim3 IgV domain matches well to that of the publishedhTim3 structure (PDB: 5F71;worldwideweb.rcsb.org/pdb/explore/explore.do?pdbld=5F71), as well as tomTim3 structure (PDB: 3KAA;worldwideweb.rcsb.org/pdb/explore/explore.do?structureld=3kaa; Rosemarieet al. (2010) J Immunol 184:1918) that was resolved in complex with PS.The PS binding pocket in hTim3 was inferred from these structuralalignments. Additionally, the location of the PS binding pocket isconserved among the TIM members in human and mouse (Freemen et al.(2010) Immunol Rev. 235: 172).

The contact residues for TIM3.18 on the hTim3 protein were identified bycalculating the difference in accessible surface area between thehTIM3:TIM3.18 Fab crystal structure and hTIM3 structure alone (“surfaceburial method”). hTIM3 residues that show buried surface area uponcomplex formation with TIM3.18 Fab were defined as being part of thecontact residues. The solvent-accessible surface of a protein wasdefined as the locus of the center of a probe sphere (representing asolvent molecule of 1.4-Å radius) as it rolls over the Van der Waalssurface of the protein. The solvent-accessible surface area wascalculated by generating surface points on an extended sphere about eachatom (at a distance from the atom center equal to the sum of the atomand probe radii), and eliminating those that lied within equivalentspheres associated with neighboring atoms as implemented in the programAREAIMOL(http://www.ccp4.ac.uk/newsletters/newsletter38/03_surfarea.html).

The results, which are shown in FIGS. 35 and 36, provide that thefollowing amino acids are contact residues, as identified by the abovedescribed surface burial method: P29, V30, C31, P38, V39, F40, E41, C42,G43, N44, V45, V46, L47, R48, T49, D50, E51, D53, R90, Q92, G95, I96,M97, D99 (numbering according to SEQ ID NO: 290, which is the maturehTIM3 extracellular domain) or P50, V51, C52, P59, V60, F61, E62, C63,G64, N65, V66, V67, L68, R69, T70, D71, E72, D74, R111, Q113, G116,I117, M118, D120 (numbering per SEQ ID NO: 286 (FIG. 20), which is hTIM3with a signal peptide). These results indicate that the contact residuesof TIM3.18.IgG1.3 on human TIM3 overlap with the PS binding pocket onhuman TIM3. Specifically, the heavy chain CDR2 of TIM3.18 occupies thePS binding pocket. Additional contacts with the PS binding loops aremade by heavy chain CDR1 and CDR3. The structural data generated hereconfirms the results obtained in the PS blocking assay (see Examples).

The crystallography results also show that the following amino acidresidues of hTIM3 have an atom that is located within 5 Å of an atom ofan amino acid residue (the “5 Å distance method”) of the TIM3.18 Fab:P29, V30, C31, P38, V39, F40, E41, C42, G43, N44, V45, V46, L47, R48,D50, E51, D53, R90, I91, Q92, G95, I96, M97, D99 (numbering according toSEQ ID NO: 290, which is the mature hTIM3 extracellular domain) or P50,V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71,E72, D74, R111, I112, Q113, G116, I117, M118, D120 (numbering per SEQ IDNO: 286 (FIG. 20), which is hTIM3 with a signal peptide). The specificinteracting residues of the Fab and hTIM3 protein are set forth in Table3.

TABLE 3 Listing of human TIM3 residues interacting with Fab residuesTim3.18 Antibody Tim3 heavy/light residue residue residue chain # type29(PRO) H/ 56 (SER) H/ 57 (GLY) H/ 58 (PHE) 30(VAL) H/ 56 (SER) H/ 58(PHE) 31(CYS) H/ 58 (PHE) H/ 55 (TYR) 38(PRO) H/ 106 (TYR) 39(VAL) H/ 60(TYR) H/ 106 (TYR) L/ 92 (TYR) L/ 93 (GLY) L/ 94 (SER) L/ 95 (SER)40(PHE) H/ 49 (TRP) H/ 54 (TYR) H/ 60 (TYR) H/ 106 (TYR) H/ 108 (HIS) L/95 (SER) L/ 97 (ILE) 41(GLU) H/ 54 (TYR) H/ 55 (TYR) H/ 56 (SER) H/ 58(PHE) H/ 60 (TYR) H/ 103 (TYR) 42(CYS) H/ 103 (TYR) H/ 104 (GLY) 43(GLY)H/ 103 (TYR) H/ 104 (GLY) H/ 105 (ASP) H/ 106 (TYR) 44(ASN) H/ 103 (TYR)H/ 104 (GLY) H/ 105 (ASP) 45(VAL) H/ 32 (ARG) H/ 33 (SER) H/ 55 (TYR) H/103 (TYR) H/ 104 (GLY) 46(VAL) H/ 32 (ARG) 47(LEU) H/ 32 (ARG) H/ 55(TYR) 48(ARG) H/ 30 (SER) H/ 31 (SER) H/ 32 (ARG) H/ 55 (TYR) 50(ASP) H/55 (TYR) H/ 56 (SER) 51(GLU) H/ 30 (SER) 53(ASP) H/ 32 (ARG) 90(ARG) H/58 (PHE) H/ 60 (TYR) 91(ILE) H/ 58 (PHE) 92(GLN) H/ 57 (GLY) H/ 58 (PHE)H/ 59 (THR) H/ 60 (TYR) 95(GLY) H/ 66 (LYS) 96(ILE) H/ 66 (LYS) 97(MET)H/ 66 (LYS) 99(ASP) H/ 58 (PHE) H/ 60 (TYR)

A comparison of the amino acid residues identified by both methods showsthat the residues are essentially the same, except for residue T49 thatis identified only by the surface burial method, and residue I91 that isidentified only by the “5 Å distance method.”

Example 18: Additional Characteristics of Tim3.18.IgG1.3

Biophysical characteristics of TIM3.18.IgG1.3 expressed in CHO cells areprovided in Table 4.

TABLE 4 Biophysical Characteristics of TIM3.18.IgG1.3 Property MethodResults Identity LC-MS/MS peptide map Deglycosylated MW = 145,619 Da (aspredicted) Deglycosylated, reduced and alkylated HC = 50,068 DaAglycosylated, reduced and alkylated LC = 23,683 Da >99% sequence anddisulfide structure confirmed by Peptide mapping and mass specPurity/Homogeneity CE-SDS 95.1% monomer, impurities include 2.9% HHL,0.9% HL, 0.5% HH, 0.6% LC; Non-glycosylated heavy chain 2.1% SEC 98.5%monomer SE-MALS 99.94% (150 kDa), 0.06% (322 kDa) HIC-HPLC 92% mainpeak, 1% pre-main peak, 7% post main peak CE (Glycans) GOF (79.3%), G1F(12.2%), G2F (0.7%), Man5 (6.8%), G0 (0.9%). cIEF Main peak pI = 8.6, pIrange 8.17-8.66 Chemical Modifications LC-MS/MS peptide map Very lowThermal Stability DSC (diluted into storage Tm1 = 68.1° C., Tm2 = 80.3°C., Tm3 = 82.6° C. and Reversibility buffer) Reversibility at 74° C. =96%, at 80° C. = 26%

A single N glycosylation site was confirmed at N297 on the heavy chain,with a glycan profile that is consistent with the glycan profile ofCHO-expressed IgG1 monoclonal antibodies. TIM3.18.IgG1.3 does not bindto CD16, CD32, or CD64, suggesting that it is inert to any Fc-FcRmediated effector function. TIM3.18.IgG1.3 has good thermal stability(Tm1=68.1° C., Tm2=80.3° C., Tm3=82.6° C.) and thermal reversibility(95.6% at 74° C., 25.5% at 80° C.), which suggest that the moleculeretains its structural integrity under thermal stress and has robustrefolding properties when stress is released.

Stability characteristics of TIM3.18.IgG1.3 are provided in Table 5.

TABLE 5 Stability of TIM3.18.IgG1.3 Property Method(s) ResultsFreeze/Thaw UV, SEC No freeze/thaw stability risk revealed (1 h @ −80°C., 1 h @ RT × 6) Solubility/Concentration Profile UV, SEC At least 60mg/mL Accelerated Stability SEC, DLS, HIC, cIEF, 12 w @ 40° C. =2%/month increase in LMW 50 mg/mL 12 w @ 4° C., 25° C., and 40° C.LC-MS/MS peptide 12 w @ 40° C. < 1% increase in HMW in the platformformulation mapping 12 w @ 40° C. = 18%/month increase in acidicvariants

No physical stability issues were observed during freeze-thaw stress (6cycles) at 50 mg/mL. Forced degradation studies at 50 mg/mL were set upat 4, 25, and 40° C. No chemical modifications in the CDR region wereobserved over 12 weeks under any condition tested.

The potential immunogenicity risk of TIM3.18.IgG1.3 was evaluated by insilico methods. The in silico iDAB analysis of TIM3.18.IgG1.3 showed fewpotential HLA binding sequences in the CDRs of this mAb, indicating alow risk of inducing a human immune response.

Example 19: PK/PD of TIM3.18.IgG1.3 in Monkeys

In a single-dose PK/PD and tolerability study, all monkeys wereimmunized intramuscularly with 2.5 mg of keyhole limpet hemocyanin (KLH)and nonproliferative recombinant adenovirus-5 (Ad5) vectors expressingsimian immunodeficiency virus (SIV) Nef and Gag proteins (3×10⁹ of eachvector). Following immunization, monkeys were intravenously administeredTIM3.18.IgG1.3 at doses of 0 (vehicle), 0.5, 10, or 25 mg/kg (N=3/group;mixed sex). Serum samples were collected for up to 42 days for theassessment of pharmacokinetics (PK) and anti-drug antibody (ADA), andblood samples were collected for up to 42 days for assessment ofreceptor occupancy. Additional serum samples were reserved for otherexploratory endpoints including soluble TIM-3 levels.

AUC_(0-168h) was dose proportional from 0.5 to 25 mg/kg. TIM3.18.IgG1.3demonstrated a T_(1/2) of about 2 weeks and total serum clearance of0.18 mL/h/kg. Volume of distribution at the steady state ranged from 68to 84 mL/kg, suggesting that TIM3.18.IgG1.3 predominantly resides in theextracellular space (Table 6).

TABLE 6 Pharmacokinetic Parameters of TIM3.18.IgG1.3 after IVAdministration in Cynomolgus Monkeys Monkey Dose AUC (_(0-INF)) T_(1/2)CLT Vss Study number (mg/kg) (μM × h) (h) (mL/h/kg) (mL/kg) DT16095 30.5 NC NC NC NC 3 10  358 ± 90* 337 ± 91  0.19 ± 0.047 84 ± 3.8 3 251076 ± 324 321 ± 104 0.17 ± 0.053 68 ± 7.4 *Extrapolated AUC exceeded20% cutoff and ranged from 21% to 55%.

Based on PK in cynomolgus monkeys and allometric scaling, the projectedhuman total serum clearance is 0.10 mL/h/kg and Vss of 88 mL/kg. As aresult, the projected human half-life is about 26 days.

Example 20: Preliminary Cytokine Release Assay

To determine if treatment with TIM3.18.IgG1.3 poses a risk of cytokinerelease syndrome, whole blood from 16 human donors was incubated with 20μg/mL of TIM3.18.IgG1.3 or positive controls in solution. A panel of 75serum cytokines and chemokines was examined for each donor. There was noevidence of enhanced T-cell-derived cytokine or chemokine release,suggesting a low risk of cytokine release syndrome. In whole bloodassays from some donors, there was elevation of IL-1β, IL-6, IL-10,TNF-α, and G-CSF, consistent with evidence presented above that TIM-3blockade increases production of monocyte or macrophage-derivedcytokines.

Example 21: TIM3.18.IgG1.3 does not Cause Receptor Downregulation orInternalization

To determine whether 13A3 downregulates or internalizes human TIM3 onthe cell membrane when binding to it, the fluorescence quenching studyshown in FIG. 37 was conducted. The results after a 3 hour treatment,which are shown in FIG. 38, indicate that neither 13A3 antibody norvariants D101E or N60Q caused dose-dependent accumulation ofintra-cellular TIM3 antibody in activated donor CD8+ T cells, suggestingthat the antibody is not internalized.

For determining potential downregulation, activated donor CD8+ T cellswere incubated for 2 hours in the presence of various amounts of 13A3,13A3.D101.Ig1.1f, 13A3.D101E/N60Q.IgG1. If or a control antibody or noantibody, and the amount of TIM3 on the cell surface was determined. Theresults indicated that incubation with the anti-TIM3 antibodies did notdownregulate cell surface TIM3.

What is claimed:
 1. An isolated antibody, which binds to human TIM3,comprising two heavy chains and two light chains, wherein each of theheavy chains comprises a heavy chain CDR1 consisting of the amino acidsequence set forth in SEQ ID NO: 41, a heavy chain CDR2 consisting ofthe amino acid sequence set forth in SEQ ID NO: 122, and a heavy chainCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 126,and wherein each of the light chains comprises a light chain CDR1consisting of the amino acid sequence set forth in SEQ ID NO: 64, alight chain CDR2 consisting of the amino acid sequence set forth in SEQID NO: 66, and a light chain CDR3 consisting of the amino acid sequenceset forth in SEQ ID NO:
 68. 2. The isolated antibody of claim 1, whereineach of the heavy chains comprises a heavy chain variable region (VH)and each of the light chains comprises a light chain variable region(VL), wherein the VH consists of the amino acid sequence set forth inSEQ ID NO: 364 and VL consists of the amino acid sequence set forth inSEQ ID NO:
 60. 3. The isolated antibody of claim 1, wherein each of theheavy chains consists of the amino acid sequence set forth in SEQ ID NO:349 and each of the light chains consists of the amino acid sequence setforth in SEQ ID NO:
 29. 4. The isolated antibody of claim 1, whereineach of the heavy chains consists of the amino acid sequence set forthin SEQ ID NO: 351 and each of the light chains consists of the aminoacid sequence set forth in SEQ ID NO:
 29. 5. The isolated antibody ofclaim 1, wherein each of the heavy chains consists of the amino acidsequence set forth in SEQ ID NO: 353 and each of the light chainsconsists of the amino acid sequence set forth in SEQ ID NO:
 29. 6. Theisolated antibody of claim 1, wherein each of the heavy chains consistsof the amino acid sequence set forth in SEQ ID NO: 350 and each of thelight chains consists of the amino acid sequence set forth in SEQ ID NO:29.
 7. The isolated antibody of claim 1, wherein each of the heavychains consists of the amino acid sequence set forth in SEQ ID NO: 352and each of the light chains consists of the amino acid sequence setforth in SEQ ID NO:
 29. 8. The isolated antibody of claim 1, whereineach of the heavy chains consists of the amino acid sequence set forthin SEQ ID NO: 354 and each of the light chains consists of the aminoacid sequence set forth in SEQ ID NO:
 29. 9. The isolated antibody ofclaim 1, wherein the antibody is selected from the group consisting ofan IgG1, an IgG2, an IgG3, an IgG4, and a variant thereof.
 10. Theisolated antibody of claim 1, comprising an effectorless IgG1 Fc. 11.The isolated antibody of claim 2, wherein the antibody is selected fromthe group consisting of an IgG1, an IgG2, an IgG3, an IgG4, and avariant thereof.
 12. A composition comprising the antibody of claim 4,and a carrier.
 13. A composition comprising the antibody of claim 7, anda carrier.
 14. The isolated antibody of claim 4, wherein the antibodycomprises at least one disulfide bond linking the two heavy chainstogether.
 15. The isolated antibody of claim 7, wherein the antibodycomprises at least one disulfide bond linking the two heavy chainstogether.
 16. An isolated antibody, which binds to human TIM3,comprising two heavy chains and two light chains, wherein each of theheavy chains comprises the amino acid sequence set forth in SEQ ID NO:351 and each of the light chains comprises the amino acid sequence setforth in SEQ ID NO:
 29. 17. The isolated antibody of claim 16, whereineach of the heavy chains consists of the amino acid sequence set forthin SEQ ID NO: 351, and each of the light chains consists of the aminoacid sequence set forth in SEQ ID NO:
 29. 18. The isolated antibody ofclaim 17, wherein the antibody comprises at least one disulfide bondlinking the two heavy chains together.
 19. An isolated antibody, whichbinds to human TIM3, comprising two heavy chains and two light chains,wherein each of the heavy chains comprises the amino acid sequence setforth in SEQ ID NO: 352 and each of the light chains the amino acidsequence set forth in SEQ ID NO:
 29. 20. The isolated antibody of claim19, wherein each of the heavy chains consists of the amino acid sequenceset forth in SEQ ID NO: 352, and each of the light chains consists ofthe amino acid sequence set forth in SEQ ID NO:
 29. 21. The isolatedantibody of claim 20, wherein the antibody comprises at least onedisulfide bond linking the two heavy chains together.
 22. A compositioncomprising the antibody of claim 5, and a carrier.
 23. A compositioncomprising the antibody of claim 6, and a carrier.
 24. A compositioncomprising the antibody of claim 17, and a carrier.
 25. A compositioncomprising the antibody of claim 20, and a carrier.
 26. A compositioncomprising the antibody of claim 21, and a carrier.
 27. The isolatedantibody of claim 5, wherein the antibody comprises at least onedisulfide bond linking the two heavy chains together.
 28. The isolatedantibody of claim 6, wherein the antibody comprises at least onedisulfide bond linking the two heavy chains together.
 29. The isolatedantibody of claim 8, wherein the antibody comprises at least onedisulfide bond linking the two heavy chains together.
 30. A compositioncomprising the antibody of claim 8, and a carrier.